Abstract: Objective: To purify and comprehensive identify α_S1-casein from bovine caseins and prepare rabbit polyclonal antibody against α_S1-casein. Method: DEAE Sepharose Fast Flow anion exchange chromatography was used to separate and purify α_S1-casein. The physicochemical property (isoelectric point and protein content), immunological technique and mass spectrometry were used to comprehensively identify α_S1-casein. Then the α_S1-casein was obtained by dialysis and lyophilization, polyclonal antibody was prepared by immunizing New Zealand white rabbit with the purified α_S1-casein, and the specificity of polyclonal antibody was analyzed. Result: Among the four kinds of caseins, α_S1-casein had the latest peak time, the largest peak area in anion exchange chromatography and the highest position in the electrophoretic diagram. Finally, the purity of α_S1-casein was 94.26%, and the yield was 27.19%. The titers of sera from two rabbits inoculated with purified α_S1-casein for five times reached 1280000 and 320000, respectively. In addition to the slight cross reaction with soybean protein (<0.25%), the antiserum showed no cross reaction with α-Lactalbumin, β-Lactoglobulin, egg white protein and peanut protein, indicating high specificity. Conclusion: This study prepared high purity α_S1-casein and high specificity polyclonal antibody against α_S1-casein, providing a new train of thought for α_S1-casein purification and comprehensive identification, and also providing material basis for the development of immunologic method for detection of α_S1-casein.