The serine protease homologous (SPH) is an important cofactor of prophenoloxidase-activating enzyme (PPAE). The gene of SPH of Chinese mitten crab Eriocheir sinensis (EsSPH) in hemocytes was cloned and characterized using reverse transcript polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The SPH cDNA consisted of 1386 bp with an open reading frame (ORF) encoded a protein of 378 amino acids, 154 bp 5'-untranslated region, and 95 bp 3'-untranslated region. Sequence comparisons against the GenBank database showed that EsSPH deduced amino acids had an overall identity to the gene of serine protease family from 41% to 70% of 15 invertebrate species. The protein had the structural characteristics of SPH, including the conserved six cysteine residues in the N-terminal clip domain and the functional activity (His157, Asp209, Gly311) in the C-terminal serine proteinase-like domain. To analyze the role of EsSPH in an acute infection, the temporal expression of the EsSPH gene after the Aeromonas hydrophila challenge was measured by real-time RT-PCR. The EsSPH transcripts in hemocytes significantly increased at 6 h, 12 h and 48 h over time after the A. hydrophila injection. This expression pattern shows that EsSPH has the potential to defend against invading microorganisms. The mRNA transcripts of EsSPH were detected in all tissues with the highest in the hepatopancreas. Interestingly, the mRNA transcripts of EsSPH and proPO were found in ova and expressed in oosperms, suggesting that the maternal transfer of EsSPH and proPO may exit in crab, but this warrants confirmation in further research.