Four trypsin-like serine proteases, designated as Fctry1, Fctry2, Fctry3, and Fctry4, were obtained from the hepatopancreas of the Chinese shrimp, Fenneropenaeus chinensis. Three trypsin-like serine proteases, specifically Fctry1, 2, and 3 were observed to have the conserved catalytic triad (H, D, and S). Regarding Fctry4, the catalytic triad S was substituted by F, and the mature peptide was found to be inactive. Further analysis for similarities indicated that Fctry1, 2 and 4 were 89-92% identical to trypsins from Pacific white shrimp (Litopenaeus vannamei); while Fctry3 was only 42% identical to trypsins from salmon louse (Lepeophtheirus salmonis). The genomic organizations of Fctry1, 2 and 4 are also quite different from Fctry3. So, Fctry3 may be a new member of the trypsin-like serine protease family. Moreover, a semi-quantitative reverse transcription polymerase chain reaction (PCR) and quantitative real-time PCR was carried out to analyze the distribution profiles and expression patterns after they were subjected to a bacterial and viral challenge. The results showed that the four trypsin-like serine proteases were upregulated in the hepatopancreas of shrimp infected with the white spot syndrome virus (WSSV), and Fctry3 increased after a bacteria challenge. Therefore, these trypsin-like serine proteases might be involved in the innate defense reactions against different pathogens in Chinese shrimp.