To elucidate the phenomenon of protein blowthrough in electrophoretic transfer and its effects on Western blotting. Using survivin antibody (a novel inhibitor of apoptosis protein), several kinds of cancer cell lysate were analyzed by Western blotting. Comparing with the traditional transfer method, two pieces of nitrocellular membranes were applied in the transfer “sandwich”. Then these two membranes were analyzed by Western blotting under same conditions. In some electrophoretic transfer circumstances, the specific Western blotting band corresponding to survivin were detected in both of them. This revealed the phenomenon of protein blowthrough in electrophoretic transfer. The length of transfer time, electric current and the molecular mass of targeted protein all affected the effects of protein blowthrough. Protein blowthrough will lead to “blotting distortion” and affect the result of quantitative analysis and qualitative analysis in Western blotting. The result revealed an important problem which required to pay more attention to in Western blotting. And it will make some help for scientifically mastering and using this technique.