The objective of this paper was to establish a method for determination of Vero cells residual protein in hepatitis A vaccine products. Vero cell protein antigen reference to immunize rabbits was prepared, and then the antibody for the development of an ELISA kit were purified. A standard curve with determination result of the prepared reference was plotted, and the various indexes of the developed kit were verified. The titer of rabbits antisera IgG was 1: 32, and the Western blot results showed specific binding of the purified antisera IgG with protein in Vero cell protein antigen reference The optimal antibody concentration for coating was 1: 400, and the working concentration of enzyme -labeled antibody was 1: 200-400. The determination result of Vero cell protein antigen reference at a concentration of 25-800 ng/mL showed a good linear relationship to a value . The developed ELISA kit showed a good specificity, precision and accuracy. Stability test showed that the ELISA kit could be kept for 6 mo in 4°C storage. Development of ELISA could be used to detect the residual Vero cells protein content in inactivated hepatitis A vaccine.