Jeffreys was born on 9 January 1950 in Oxford, Oxfordshire, England. He came from a middle-class family and has one brother and sister. He spent the first six years of his life in Oxford until 1956 when the family moved to Luton. His curiosity and inventiveness were probably gained from his father, as well as his paternal grandfather who had a number of patents to his name. When he was eight years old his father gave him a large chemistry set which was enhanced over the next few years with extra chemicals including a bottle of concentrated sulphuric acid, bought from a pharmacy, at a time when pharmacists were less regulated than now. He liked making small explosions, but an accidental splash of sulphuric acid caused a burn and a permanent scar on his chin (now under his beard). When he was about eight or nine years old, his father bought him a beautiful Victorian brass microscope, which he used to examine biological specimens, furthering his interest in biology. At about 12 years old he made a small dissecting kit (including a scalpel crafted from a flattened pin) which he used to dissect a bumblebee, but he got into trouble with his parents when he progressed to dissecting a larger specimen. One Sunday morning he found a dead cat on the road while doing his paper round and took it home in his bag. He started to dissect it before Sunday lunch on the dining room table causing a foul smell throughout the house, which was particularly bad after he ruptured its intestines.
Jeffreys was a pupil at Luton Grammar School and then Luton Sixth Form College. He followed the youth culture of the time and initially became a Mod while owning a Vespa 150 cc motor-scooter and wearing a parka jacket. He was then a Hippie for a while before buying a Matchless 350 cc motorcycle and becoming a Rocker. From 1968 he was a student at Merton College, Oxford and in 1972 he graduated with first-class honours degree in biochemistry.
Jeffreys enjoys being at the laboratory bench, and prepared his PhD thesis entitled "Studies on the mitochondria of cultured mammalian cells" as a postgraduate student at the Genetics Laboratory, University of Oxford. After finishing his PhD, he moved to the University of Amsterdam, where he worked on mammalian genes as a research fellow. He moved on to the University of Leicester in 1977, where he found an academically stimulating and helpful environment. Working in Leicester he discovered a method of showing variations between individual's DNA in 1984, and invented and developed genetic fingerprinting.
Jeffreys had a "eureka moment" in his lab in Leicester after looking at the X-ray of a DNA experiment at 9:05 am on Monday 10 September 1984, which unexpectedly showed both similarities and differences in his technician's family's DNA. Within about half an hour, he realized the possible scope of DNA fingerprinting, which uses variations in the genetic code to identify individuals. The method has become important in forensic science to assist police detective work, and it has also proved useful in resolving paternity and immigration disputes. The method can also be applied to non-human species, for example in wildlife population genetics studies. Before his methods were commercialised in 1987 his laboratory was the only centre carrying out DNA fingerprinting in the world, and during this period of about two or three years it was very busy, receiving inquiries from all over the globe.
Jeffreys' DNA method, which is often called DNA fingerprinting, was first put to use when he was asked to help in a disputed immigration case to confirm the identity of a British boy whose family was originally from Ghana. The case was resolved when the DNA results proved that the boy was closely related to the other members of the family, and Jeffreys saw the relief in the mother's face when she heard the results. DNA fingerprinting was first used as a police forensic test to identify the rapist and killer of two teenagers, Lynda Mann and Dawn Ashworth, who were both murdered in Narborough, Leicestershire, in 1983 and 1986 respectively. Colin Pitchfork was identified and convicted of murder after samples taken from him matched semen samples taken from the two dead girls. This turned out to be a specifically important identification for without it, British Authorities believe that Richard Buckland, the main suspect, would have inevitably been convicted. Therefore, not only did Jeffrey's work in this case prove who the real killer was, but exonerate someone who likely would have spent his life in prison otherwise. In 1992, Jeffreys' methods were used to confirm the identity for German prosecutors of the Nazi Dr. Josef Mengele, who had died in 1979, by comparing DNA obtained from a femur bone of his exhumed skeleton, and DNA from his widow and son, in a similar way to paternity testing.