Mullis was born in Lenoir, North Carolina, near the Blue Ridge Mountains, on December 28, 1944. His family had a background in farming in this rural area. As a child, Mullis recalls, he was interested in observing organisms in the countryside. He grew up in Columbia, South Carolina, where he attended Dreher High School. He has described his early interest in chemistry, and claims to have learned how to chemically synthesize and build solid state fuel propulsion rockets as a high school student during the 1950s.

Mullis earned a Bachelor of Science (BS) degree in chemistry from the Georgia Institute of Technology in Atlanta in 1966, during which time he got married and started a Business. He then received a PhD in biochemistry from the University of California, Berkeley in 1973; his research done in J. B. Neilands' laboratory focused on synthesis and structure of bacterial iron transporter molecules. Following his graduation, Mullis became a postdoctoral fellow in pediatric cardiology at the University of Kansas Medical School, going on to complete two years of postdoctoral work in pharmaceutical chemistry at the University of California, San Francisco.

The suggestion that Mullis was solely responsible for the idea of using Taq polymerase in the PCR process has been contested by his co-workers at the time, who were embittered by his abrupt departure from Cetus. However, other Scientists have written that "the full potential [of PCR] was not realized" until Mullis' work in 1983, and that Mullis' colleagues failed to see the potential of the technique when he presented it to them. As a result, some controversy surrounds the balance of credit that should be given to Mullis versus the team at Cetus. In practice, credit has accrued to both the Inventor and the company (although not its individual workers) in the form of a Nobel Prize and a $10,000 Cetus bonus for Mullis and $300 million for Cetus when the company sold the patent to Roche Molecular Systems. After DuPont lost out to Roche on that sale, the company unsuccessfully disputed Mullis's patent on the alleged grounds that PCR had been previously described in 1971. Mullis and Erlich took Cetus' side in the case, and Khorana refused to testify for DuPont; the jury upheld Mullis's patent in 1991.. However later, in February 1999, the patent of Hoffman-La Roche (United States Patent No. 4,889,818) was found by the courts to be unenforceable, after Dr. Thomas Kunkel testified in the case "Hoffman-La Roche v. Promega Corporation" on behalf of the defendants (Promega Corporation) that "prior art" (i.e. articles on the subject of polymerase chain reaction, published by other groups, prior to the work of Mullis and the patent application) existed, in the form of two articles, published by Alice Chien et al. in 1976 , and A. S. Kaledin et al. in 1980 .

Mullis enjoys surfing. He has been married four times. He has three children by two ex-wives. Mullis reported an encounter with a "glowing green raccoon" at his cabin in the woods of northern California around midnight one night in 1985; he denies having been on LSD or any other drug at the time.

A complication at that point was that the DNA polymerase used was destroyed by the high heat used at the start of each replication cycle and had to be replaced. In 1986, Saiki started to use Thermophilus aquaticus (Taq) DNA polymerase to amplify segments of DNA. The Taq polymerase was heat resistant and would only need to be added once, thus making the technique dramatically more affordable and subject to automation. This has created revolutions in biochemistry, molecular biology, genetics, Medicine and forensics.

Mullis also received the John Scott Award in 1991, given by the City Trusts of Philadelphia to others including Thomas Edison and the Wright Brothers.

In 1992, Mullis founded a Business with the intent to sell pieces of jewelry containing the amplified DNA of deceased famous people like Elvis Presley and Marilyn Monroe. Mullis is also a member of the USA Science and Engineering Festival's Advisory Board. As of 2014, he is a researcher at the Children's Hospital Oakland Research Institute in Oakland, California.

Mullis details his experiences synthesizing and testing various psychedelic amphetamines and a difficult trip on DET in his autobiography. In a Q&A interview published in the September 1994 issue of California Monthly, Mullis said, "Back in the 1960s and early '70s I took plenty of LSD. A lot of people were doing that in Berkeley back then. And I found it to be a mind-opening experience. It was certainly much more important than any courses I ever took." During a symposium held for centenarian Albert Hofmann, "Hofmann revealed that he was told by Nobel-prize-winning Chemist Kary Mullis that LSD had helped him develop the polymerase chain reaction that helps amplify specific DNA sequences."

The Anthropologist Paul Rabinow wrote a book on the history of the PCR method in 1996 (entitled Making PCR) in which he discussed whether or not Mullis "invented" PCR or "merely" came up with the concept of it. Rabinow, a Foucault scholar interested in issues of the production of knowledge, used the topic to argue against the idea that scientific discovery is the product of individual work, writing, "Committees and science journalists like the idea of associating a unique idea with a unique person, the lone genius. PCR is thought by some to be an Example of teamwork, but by others as the genius of one who was smart enough to put things together which were present to all, but overlooked. For Mullis, the light bulb went off, but for others it did not. This is consistent with the idea, that the prepared (educated) mind who is careful to observe and not overlook, is what separates the genius scientist from his many also smart Scientists. The proof is in the fact that the person who has the light bulb go off never forgets the Ah experience, while the others never had this photochemical reaction go off in their brains."

Mullis's 1998 autobiography Dancing Naked in the Mind Field, gives his account of the commercial development of PCR, as well as providing insights into his opinions and experiences. In the book, Mullis chronicles his romantic relationships, use of LSD, synthesis and self-testing of novel psychoactive substances, belief in astrology and an encounter with an extraterrestrial in the form of a fluorescent raccoon.

Mullis has also invented a UV-sensitive plastic that changes color in response to light, and most recently has been working on an approach for mobilizing the immune system to neutralize invading pathogens and toxins, leading to the formation of his current venture, Altermune LLC, in 2011. Mullis described this idea this way:

Other Cetus Scientists, including Randall Saiki and Henry Erlich, were placed on PCR projects to work on evaluating whether PCR could amplify a specific human gene (betaglobin) from genomic DNA. Saiki generated the needed data and Erlich authored the first paper to include utilization of the technique, while Mullis was still working on a paper that would describe PCR itself. Mullis’s 1985 paper with R. K. Saiki and H. A. Erlich, “Enzymatic Amplification of β-globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia” -- the polymerase chain reaction invention (PCR) -- was honored by a Citation for Chemical Breakthrough Award from the Division of History of Chemistry of the American Chemical Society in 2017.