Beginning at age 16 in the Preparatory Department, Morgan attended the State College of Kentucky (now the University of Kentucky). He focused on science; he particularly enjoyed natural history, and worked with the U.S. Geological Survey in his summers. He graduated as valedictorian in 1886 with a Bachelor of Science degree. Following a summer at the Marine Biology School in Annisquam, Massachusetts, Morgan began graduate studies in zoology at the recently founded Johns Hopkins University, the first research-oriented American university. After two years of experimental work with morphologist william Keith Brooks and writing several publications, Morgan was eligible to receive a master of science from the State College of Kentucky in 1888. The college required two years of study at another institution and an examination by the college faculty. The college offered Morgan a full professorship; however, he chose to stay at Johns Hopkins and was awarded a relatively large fellowship to help him fund his studies.

In 1890, Morgan was appointed associate professor (and head of the biology department) at Johns Hopkins' sister school Bryn Mawr College, replacing his colleague Edmund Beecher Wilson. Morgan taught all morphology-related courses, while the other member of the department, Jacques Loeb, taught the physiological courses. Although Loeb stayed for only one year, it was the beginning of their lifelong friendship. Morgan lectured in biology five days a week, giving two lectures a day. He frequently included his own recent research in his lectures. Although an enthusiastic Teacher, he was most interested in research in the laboratory. During the first few years at Bryn Mawr, he produced descriptive studies of sea acorns, ascidian worms and frogs.

In 1894 Morgan was granted a year's absence to conduct research in the laboratories of Stazione Zoologica in Naples, where Wilson had worked two years earlier. There he worked with German Biologist Hans Driesch, whose research in the experimental study of development piqued Morgan's interest. Among other projects that year, Morgan completed an experimental study of ctenophore embryology. In Naples and through Loeb, he became familiar with the Entwicklungsmechanik (roughly, "developmental mechanics") school of experimental biology. It was a reaction to the vitalistic Naturphilosophie, which was extremely influential in 19th-century morphology. Morgan changed his work from traditional, largely descriptive morphology to an experimental embryology that sought physical and chemical explanations for organismal development.

When Morgan returned to Bryn Mawr in 1895, he was promoted to full professor. Morgan's main lines of experimental work involved regeneration and larval development; in each case, his goal was to distinguish internal and external causes to shed light on the Roux-Driesch debate. He wrote his first book, The Development of the Frog's Egg (1897). He began a series of studies on different organisms' ability to regenerate. He looked at grafting and regeneration in tadpoles, fish and earthworms; in 1901 he published his research as Regeneration.

In 1900 three Scientists, Carl Correns, Erich von Tschermak and Hugo De Vries, had rediscovered the work of Gregor Mendel, and with it the foundation of genetics. De Vries proposed that new species were created by mutation, bypassing the need for either Lamarckism or Darwinism. As Morgan had dismissed both evolutionary theories, he was seeking to prove De Vries' mutation theory with his experimental heredity work. He was initially skeptical of Mendel's laws of heredity (as well as the related chromosomal theory of sex determination), which were being considered as a possible basis for natural selection.

In the following years, most biologists came to accept the Mendelian-chromosome theory, which was independently proposed by Walter Sutton and Theodor Boveri in 1902/1903, and elaborated and expanded by Morgan and his students. Garland Allen characterized the post-1915 period as one of normal science, in which "The activities of 'geneticists' were aimed at further elucidation of the details and implications of the Mendelian-chromosome theory developed between 1910 and 1915." But, the details of the increasingly complex theory, as well as the concept of the gene and its physical nature, were still controversial. Critics such as W. E. Castle pointed to contrary results in other organisms, suggesting that genes interact with each other, while Richard Goldschmidt and others thought there was no compelling reason to view genes as discrete units residing on chromosomes.

Morgan was interested in evolution throughout his life. He wrote his thesis on the phylogeny of sea spiders (pycnogonids) and wrote four books about evolution. In Evolution and Adaptation (1903), he argued the anti-Darwinist position that selection could never produce wholly new species by acting on slight individual differences. He rejected Darwin's theory of sexual selection and the Neo-Lamarckian theory of the inheritance of acquired characters. Morgan was not the only scientist attacking natural selection. The period 1875–1925 has been called 'The eclipse of Darwinism'. After discovering many small stable heritable mutations in Drosophila, Morgan gradually changed his mind. The relevance of mutations for evolution is that only characters that are inherited can have an effect in evolution. Since Morgan (1915) 'solved the Problem of heredity', he was in a unique position to examine critically Darwin's theory of natural selection.

Later in 1904, E. B. Wilson—still blazing the path for his younger friend—invited Morgan to join him at Columbia University. This move freed him to focus fully on experimental work.

Following C. W. Woodworth and william E. Castle, around 1908 Morgan started working on the fruit fly Drosophila melanogaster, and encouraging students to do so as well. With Fernandus Payne, he mutated Drosophila through physical, chemical, and radiational means. He began cross-breeding experiments to find heritable mutations, but they had no significant success for two years. Castle had also had difficulty identifying mutations in Drosophila, which were tiny. Finally in 1909, a series of heritable mutants appeared, some of which displayed Mendelian inheritance patterns; in 1910 Morgan noticed a white-eyed mutant male among the red-eyed wild types. When white-eyed flies were bred with a red-eyed female, their progeny were all red-eyed. A second generation cross produced white-eyed males—a sex-linked recessive trait, the gene for which Morgan named white. Morgan also discovered a pink-eyed mutant that showed a different pattern of inheritance. In a paper published in Science in 1911, he concluded that (1) some traits were sex-linked, (2) the trait was probably carried on one of the sex chromosomes, and (3) other genes were probably carried on specific chromosomes as well.

Morgan and his students became more successful at finding mutant flies; they counted the mutant characteristics of thousands of fruit flies and studied their inheritance. As they accumulated multiple mutants, they combined them to study more complex inheritance patterns. The observation of a miniature-wing mutant, which was also on the sex chromosome but sometimes sorted independently to the white-eye mutation, led Morgan to the idea of genetic linkage and to hypothesize the phenomenon of crossing over. He relied on the discovery of Frans Alfons Janssens, a Belgian professor at the University of Leuven, who described the phenomenon in 1909 and had called it chiasmatypie. Morgan proposed that the amount of crossing over between linked genes differs and that crossover frequency might indicate the distance separating genes on the chromosome. The later English Geneticist J. B. S. Haldane suggested that the unit of measurement for linkage be called the morgan. Morgan's student Alfred Sturtevant developed the first genetic map in 1913.

He returned to embryology and worked to encourage the spread of genetics research to other organisms and the spread of the mechanistic experimental approach (Enwicklungsmechanik) to all biological fields. After 1915, he also became a strong critic of the growing eugenics movement. This adopted the ideas of genetics in support of racism and worse.

In A Critique of the Theory of Evolution (1916), Morgan discussed questions such as: "Does selection play any role in evolution? How can selection produce anything new? Is selection no more than the elimination of the unfit? Is selection a creative force?" After eliminating some misunderstandings and explaining in detail the new science of Mendelian heredity and its chromosomal basis, Morgan concludes, "the evidence shows clearly that the characters of wild animals and plants, as well as those of domesticated races, are inherited both in the wild and in domesticated forms according to the Mendel's Law". "Evolution has taken place by the incorporation into the race of those mutations that are beneficial to the life and reproduction of the organism". Injurious mutations have practically no chance of becoming established. Far from rejecting evolution, as the title of his 1916 book may suggest, Morgan laid the foundation of the science of genetics. He also laid the theoretical foundation for the mechanism of evolution: natural selection. Heredity was a central plank of Darwin's theory of natural selection, but Darwin could not provide a working theory of heredity. Darwinism could not progress without a correct theory of genetics. By creating that foundation, Morgan contributed to the neo-Darwinian synthesis, despite his criticism of Darwin at the beginning of his career. Much work on the Evolutionary Synthesis remained to be done.

In accordance with his reputation, Morgan held numerous prestigious positions in American science organizations. From 1927 to 1931 Morgan served as the President of the National Academy of Sciences; in 1930 he was the President of the American Association for the Advancement of Science; and in 1932 he chaired the Sixth International Congress of Genetics in Ithaca, New York. In 1933 Morgan was awarded the Nobel Prize in Physiology or Medicine; he had been nominated in 1919 and 1930 for the same work. As an acknowledgement of the group nature of his discovery he gave his prize money to Bridges', Sturtevant's and his own children. Morgan declined to attend the awards ceremony in 1933, instead attending in 1934. The 1933 rediscovery of the giant polytene chromosomes in the salivary gland of Drosophila may have influenced his choice. Until that point, the lab's results had been inferred from phenotypic results, the visible polytene chromosome enabled them to confirm their results on a physical basis. Morgan's Nobel acceptance speech entitled "The Contribution of Genetics to Physiology and Medicine" downplayed the contribution genetics could make to Medicine beyond genetic counselling. In 1939 he was awarded the Copley Medal by the Royal Society.

Morgan moved to California to head the Division of Biology at the California Institute of Technology in 1928. In establishing the biology division, Morgan wanted to distinguish his program from those offered by Johns Hopkins and Columbia, with research focused on genetics and evolution; experimental embryology; physiology; biophysics and biochemistry. He was also instrumental in the establishment of the Marine Laboratory at Corona del Mar. He wanted to attract the best people to the Division at Caltech, so he took Bridges, Sturtevant, Jack Shultz and Albert Tyler from Columbia and took on Theodosius Dobzhansky as an international research fellow. More Scientists came to work in the Division including George Beadle, Boris Ephrussi, Edward L. Tatum, Linus Pauling, Frits Went, and Sidney W. Fox.

He received two extensions of his contract at Caltech, but eventually retired in 1942, becoming professor and chairman emeritus. George Beadle returned to Caltech to replace Morgan as chairman of the department in 1946. Although he had retired, Morgan kept offices across the road from the Division and continued laboratory work. In his retirement, he returned to the questions of sexual differentiation, regeneration, and embryology.

Morgan had throughout his life suffered with a chronic duodenal ulcer. In 1945, at age 79, he experienced a severe heart attack and died from a ruptured artery.

Under Brooks, Morgan completed his thesis work on the embryology of sea spiders—collected during the summers of 1889 and 1890 at the Marine Biological Laboratory in Woods Hole, Massachusetts—to determine their phylogenetic relationship with other arthropods. He concluded that with respect to embryology, they were more closely related to spiders than crustaceans. Based on the publication of this work, Morgan was awarded his Ph.D. from Johns Hopkins in 1890, and was also awarded the Bruce Fellowship in Research. He used the fellowship to travel to Jamaica, the Bahamas and to Europe to conduct further research.