Abstract: We cloned the Stefin gene of grass carp which was 294 bp, encoding a mature polypeptide of 97 amino acids lacking of disulfide bond and containing the typical conserved domain of Stefin (family Ⅰ), such as Gly (3 and 4) and QXVXG(45~49). Homology analysis indicates that grass carp Stefin A shared the highest amino acid sequence identity of 47.5% with Burton's mouthbrooder (Haplochromis burtoni) Stefin A1. Phylogenetic tree analysis indicats that grass carp Stefin A held together with Burton's mouthbrooder, Colisa chuna (Trichogaster chuna), lamprologini (Neolamprologus brichard), elephant shark (Callorhinchus milii) and bicolor damselfish (Stegastes partitus). Recombinant Stefin was expressed by 1 mol·L^-1 IPTG and the target protein was gradiently washed by urea and purified by Ni²⁺-NTA agarose affinity chromatography. SDS-PAGE and HPLC of TSK-GEL G2000SWxl were conducted to examine the results of expression and purification. The purified protein appeared as a single band on the SDS-PAGE, corresponding to a molecular weight of approximately 11.4 kD. And it also appeared as a single active peak on TSK-GEL G2000SWxl with purity of 96.28%. The activity assay (Z-Phe-Arg-MCA as a substrate) was finally performed to characterize the inhibitory effect of the recombinant Stefin to Cathepsin B and Cathepsin L from carp. The results reveal that it can inhibit the activities of these two proteinases effectively.