Plasmin plays a significant role in cheese ripening and characteristics of dairy products. The antibiotic could be presented in milk as residuals from animal treated by antibiotic, while presence of detergent could be as residuals from cleaning of milking machines, tanks during transpirations, storage tanks and pasteurization equipments etc. The effect of detergents and antibiotic residuals on proteolytic action of plasmin on β-casein in model solution was determined by Urea-polyacrylamide gel electrophoresis. The results suggested that plasmin followed the proteolytic pathway on cow b-casein to produce g1, g2 and g3 as the degradation fragments. The proteolytic pathway has been affected by presence of sodium hydroxide and nitric acid, while was not affected by presence of antibiotic. The results also indicated that the presence of sodium hydrioxid increased the plasmin activity, while plasmin hydrolysate of B-casein slightly inhibited by nitric acid. The proteolytic activity of plasmin on b-casein was broader and higher at the end of incubation period, to yield low-sized fragments as evidenced with polyacrylamide gel electrophoresis. Increased plasmin activity by sodium hydroxide residual could affect cheese making characteristics such as the rennet coagulation time, curd strength, and curd syneresis, produce off-flavor and bitterness in cheese. Since the plasmin is heat stable enzyme, increasing its activity in UHT milk could case gelatoin and produce off-flavor and bitterness.