Concerned that he might not be able to afford to go to a university, Wilson tried to enlist in the United States Army. He planned to earn U.S. government financial support for his education, but failed the Army medical examination due to his impaired eyesight. Wilson was able to afford to enroll in the University of Alabama after all, earning his B.S. and M.S. degrees in biology there in 1950. In 1952 he transferred to Harvard University.

Appointed to the Harvard Society of Fellows, he could travel on overseas expeditions, collecting ant species of Cuba and Mexico and travel the South Pacific, including Australia, New Guinea, Fiji, New Caledonia and Sri Lanka. In 1955, he received his Ph.D. and married Irene Kelley.

From 1956 until 1996 Wilson was part of the faculty of Harvard. He began as an ant taxonomist and worked on understanding their evolution, how they developed into new species by escaping environmental disadvantages and moving into new habitats. He developed a theory of the "taxon cycle".

Wilson has said that, if he could start his life over he would work in microbial ecology, when discussing the reinvigoration of his original fields of study since the 1960s. He studied the mass extinctions of the 20th century and their relationship to modern society, and in 1998 argued for an ecological approach at the Capitol:

In 1971, he published the book The Insect Societies about the biology of social insects like ants, bees, wasps and termites. In 1973, Wilson was appointed 'Curator of Insects' at the Museum of Comparative Zoology. In 1975, he published the book Sociobiology: The New Synthesis applying his theories of insect behavior to vertebrates, and in the last chapter, humans. He speculated that evolved and inherited tendencies were responsible for hierarchical social organisation among humans. In 1978 he published On Human Nature, which dealt with the role of biology in the evolution of human culture and won a Pulitzer Prize for General Nonfiction.

Marshall Sahlins's 1976 work The Use and Abuse of Biology was a direct criticism of Wilson's theories.

Wilson wrote in his 1978 book On Human Nature, "The evolutionary epic is probably the best myth we will ever have." Wilson's use of the word "myth" provides people with meaningful placement in time celebrating shared heritage. Wilson's fame prompted use of the morphed phrase epic of evolution. The book won the Pulitzer Prize in 2011.

Wilson is the Pellegrino University Research Professor, Emeritus in Entomology for the Department of Organismic and Evolutionary Biology at Harvard University, a lecturer at Duke University, and a Fellow of the Committee for Skeptical Inquiry. He is a Humanist Laureate of the International Academy of Humanism. He is a two-time winner of the Pulitzer Prize for General Nonfiction (for On Human Nature in 1979, and The Ants in 1991) and a New York Times bestseller for The Social Conquest of Earth, Letters to a Young Scientist, and The Meaning of Human Existence.

In 1981 after collaborating with Charles Lumsden, he published Genes, Mind and Culture, a theory of gene-culture coevolution. In 1990 he published The Ants, co-written with Bert Hölldobler, his second Pulitzer Prize for General Nonfiction.

Objections from evangelical Christians included those of Paul E. Rothrock in 1987: "... sociobiology has the potential of becoming a religion of scientific materialism." Philosopher Mary Midgley encountered Sociobiology in the process of writing Beast and Man (1996) and significantly rewrote the book to offer a critique of Wilson's views. Midgley praised the book for the study of animal behavior, clarity, scholarship, and encyclopedic scope, but extensively critiqued Wilson for conceptual confusion, scientism, and anthropomorphism of genetics.

Wilson, along with Bert Hölldobler, carried out a systematic study of ants and ant behavior, culminating in the 1990 encyclopedic work The Ants. Because much self-sacrificing behavior on the part of individual ants can be explained on the basis of their genetic interests in the survival of the sisters, with whom they share 75% of their genes (though the actual case is some species' queens mate with multiple males and therefore some workers in a colony would only be 25% related), Wilson argued for a sociobiological explanation for all social behavior on the model of the behavior of the social insects.

In 1996, Wilson officially retired from Harvard University, where he continues to hold the positions of Professor Emeritus and Honorary Curator in Entomology. He founded the E.O. Wilson Biodiversity Foundation, which finances the PEN/E. O. Wilson Literary Science Writing Award and is an "independent foundation" at the Nicholas School of the Environment, Duke University. Wilson became a special lecturer at Duke University as part of the agreement.

Understanding the scale of the extinction crisis has led him to advocate for forest protection, including the "Act to Save America's Forests", first introduced in 1998, until 2008, but never passed. The Forests Now Declaration calls for new markets-based mechanisms to protect tropical forests. In 2014, Wilson called for setting aside 50% of the earth's surface for other species to thrive in as the only possible strategy to solve the extinction crisis.

Wilson has published 14 books during the new millennium: The Future of Life, 2002, Pheidole in the New World: A Dominant, Hyperdiverse Ant Genus, 2003, From So Simple a Beginning: Darwin's Four Great Books, 2005, The Creation: An Appeal to Save Life on Earth, September 2006, Nature Revealed: Selected Writings 1949–2006, The Superorganism: The Beauty, Elegance, and Strangeness of Insect Societies, 2009, Anthill: A Novel April 2010, Kingdom of Ants: Jose Celestino Mutis and the Dawn of Natural History in the New World, 2010, The Leafcutter Ants: Civilization by Instinct, 2011, The Social Conquest of Earth, 2012, Half-Earth: Our Planet's Fight for Life, 2016.

Wilson coined the phrase scientific humanism as "the only worldview compatible with science's growing knowledge of the real world and the laws of nature". Wilson argued that it is best suited to improve the human condition. In 2003, he was one of the signers of the Humanist Manifesto.

Sociobiology was initially met with substantial criticism. Several of Wilson's colleagues at Harvard, such as Richard Lewontin and Stephen Jay Gould, were strongly opposed to his ideas regarding sociobiology. Gould, Lewontin, and others from the Sociobiology Study Group from the Boston area wrote "Against 'Sociobiology'" in an open letter criticizing Wilson's "deterministic view of human society and human action". Although attributed to members of the Sociobiology Study Group, it seems that Lewontin was the main author. In a 2011 interview, Wilson said, "I believe Gould was a charlatan. I believe that he was ... seeking reputation and credibility as a scientist and Writer, and he did it consistently by distorting what other Scientists were saying and devising arguments based upon that distortion."

He published 3 books in 2014 alone: Letters to a Young Scientist, A Window on Eternity: A Biologist's Walk Through Gorongosa National Park, and The Meaning of Human Existence.

In a New Scientist interview published on 21 January 2015, Wilson said that "Religion 'is dragging us down' and must be eliminated 'for the sake of human progress'", and "So I would say that for the sake of human progress, the best thing we could possibly do would be to diminish, to the point of eliminating, religious faiths." On the question of God, Wilson has described his position as provisional deism and explicitly denied the label of "atheist", preferring "agnostic". He has explained his faith as a trajectory away from traditional beliefs: "I drifted away from the church, not definitively agnostic or atheistic, just Baptist & Christian no more." Wilson argues that the belief in God and rituals of religion are products of evolution. He argues that they should not be rejected or dismissed, but further investigated by science to better understand their significance to human nature. In his book The Creation, Wilson suggests that Scientists ought to "offer the hand of friendship" to religious Leaders and build an alliance with them, stating that "Science and religion are two of the most potent forces on Earth and they should come together to save the creation."

The book was mentioned in Jonathan Haidt’s book The Righteous Mind.