It has been proved that natural immunity played an vital role in invertebrate animals, and mainly depended on nonspecific immunit y molecules with the mode of pathogenassociated molecular pattern to recognize pathogen and then to perform immune functions. In order to search the main pro teins with function of recognizing directly pathogen in shrimp serum, an attempt was made to purify and identify the proteins bound directly to pathogens includ ing Vibrio alginolyticus,Vibrio harveyi, Aeromonas hydrophila an d Staphylococcus aureus in shrimp Litopenaeus vannamei serum using the methods of affinity proteomic and Westernblotting in th e current study. The results showed that a protein with 75 000 (namely p75) in the shrimp serum could bind directly to the four pathogens in comparison wit h its control group respectively after the four types of pathogen reacted with shri mp serum for 5 h. While they reacted for 15 h another shrimp serum protein with 77 000 (namely p77) and several other proteins (0-4) with different molecular we ights also bound directly to the four pathogens. Then p75 and p77 proteins were cut out from the gels and subjected to MALDI-TOF/MS analysis. The search results obtained from Mascot search engine in NCBInr database indicated that p75 and p7 7 proteins were identified to be Litopenaeus vannimei hemocyanin sub units with about 75 000 and 77 000 respectively. Moreover, the p75 protein was d ocumented to be strongly reacted with rabbit anti-shrimp hemocyanin 75 000 subun it antibody specially. Thus, these results led to be deduced that hemocyanin was the main protein binding directly to different pathogens in the shrimp L. vannimei serum, suggesting that hemocyanin probably possesses directly a ntibacterial function and would be helpful for research of hemocyanin’s nonsp ecific immune activities and its mechanism.