A conjugated linoleic acid isomer biological enrichment method includes preparation of a biological enrichment culture medium, preparation of production strains, conjugated linoleic acid isomer biological enrichment and other steps, and c9,t11-conjugated linoleic acid (CLA) isomers can be obtained. The c9,t11-conjugated linoleic acid (CLA) isomer content is greater than 0.45% by mass, substrate conversion rate is more than 90%, the c9,t11-conjugated linoleic acid (CLA) isomer content in products is increased significantly, by biological enrichment culture, not only an anti-cancer function product rich in the c9,t11-conjugated linoleic acid (CLA) isomer can be obtained, proteins and celluloses in defatted soybean also can be decomposed into polypeptides and soluble dietary fiber and other health care function active factors, the health care function of the products is significantly enhanced, and the application range of the product is further broadened.