The invention relates to a biological enrichment method of a conjugated linolenic acid isomer. The biological enrichment method comprises biological enrichment culture medium preparation, production strain preparation, biological enrichment of the conjugated linolenic acid isomer, and other steps to obtain the c9,t11,c15-conjugated linolenic acid isomer. According to the present invention, the c9,t11,c15-conjugated linolenic acid isomer mass content in the obtained product is more than 0.36%, and the substrate conversion rate is more than 90%; and with the biological enrichment culture, the c9,t11,c15-conjugated linolenic acid isomer-rich anti-cancer function product is obtained, proteins and cellulose in defatted soybean can be decomposed into polypeptides, soluble dietary fibers and other active factors having health functions through lactic acid bacteria, and the health function of the product is significantly enhanced so as to further broaden the application range of the product.