Abstract: Objective: Research on buffalo milk affected by the systems about mixed enzyme and composite enzyme including papain, neutral protease and alkaline protease, comparing with the result in different ways of hydrolysis. Method: First, the experiment went on with the single factor of three proteases hydrolyzing milk to explore the optimum condition of enzymatic hydrolysis.On this basis, the degree of hydrolysis was determinated through composite enzymatic hydrolysis and mixed enzyme and anticoagulant activity was determinated.Consequence: The results showed that optimum condition of papain were as follows:the ratio of material to water for 1∶ 2, p H = 5.0, temperature 60 ℃, time 2 h, enzyme dosage 6000 u/m L.Neutral protease of were as follows: the ratio of material to water for 1∶ 2, p H = 6.0, temperature 40 ℃, time of 2 h, enzyme dosage 6000 u/m L.Optimum condition of alkaline protease were as follows: the ratio of material to water for 1 ∶ 2, p H = 8.0, temperature 50 ℃, time 4 h, enzyme dosage 6000 u/m L. Composite enzymatic hydrolysis anticoagulant activity was higher than by mixed enzymatic hydrolysis.The highest anticoagulant activity was 52 ATU/m L. Conclusion: In the experimental conditions, the degree and anticoagulant activity by composite enzymatic hydrolysis were higher than by mixed enzymatic hydrolysis. The enzymatic hydrolysis solution, which was prepared by the enzyme combination of the composite enzyme of neutral protease and papain protease and alkaline protease, had the highest anticoagulant activity, was 52.0 ATU/m L.