Objective: The difference in bioactive substance content and bioactivity in black beans were compared. Methods: The contents of total phenolic acids, total flavonoids, condensed tannins, total anthocyanins, total polysaccharides and total proteins of nine kinds of black beans were determined. The antioxidant activities of black bean extracts in vitro and their inhibitory effects on α-amylase, pancreatic lipase, acetylcholinesterase and tyrosinase were compared. Results: The contents of total phenolic acid, total flavonoids, condensed tannins and total anthocyanins were as follows: equine bean > small black bean > black bean; Total polysaccharide content: small black bean > black bean > horse bean; Total protein content: horse bean > black bean > small black bean. Antioxidant activity: horse bean > small black bean > black bean. In vitro experiments showed that little black bean and black bean had inhibitory effects on many enzymes, and the intensity was different. Conclusion: The contents and functions of bioactive substances in different varieties of black beans are different. Black beans are more suitable for eating, and horse bean and small black beans have higher medicinal value.