Firstly; the Tricine-SDS-PAGE and filter paper diffusion were used to identify the enzymatic hydrolysis of Silver carp recombinant Cystatin with Papain and antibacterial effect of Cystatin's hydrolyzate on the three kinds of dominant spoilage strains;Pseudomonas?fragi;Shewanella?putrefaciensandAeromonas?media. The results indicated that the hydrolyzate of 6 h group was less than 9.5 ku and its antibacterial effect was the best among the three dominant spoilage strains. Therefore; the hydrolyzate of 6 h group were purified and analyzed by HPLC with column of TSK-GEL G2000SWXL gel and two-dimensional electrophoresis. The results indicated that only the peak T-2 had significant antibacterial activity for the dominant spoilage and exhibited a single peak on HPLC and a single protein point with the molecular weight of 8.6 ku and the pI of 6.5 on 2-D gel. At last; RPLC-MS/MS was used to identify the protein spot and obtained three peptides fragments including 31QSNDAFVR38; 46VQQQVAAGMKY56 and 81NPSIEQVIQ89; which contained the characteristic sequence of Cystatin QVAAG. These results suggested that Cystatin protein with intact sequence of 12 ku-14 ku contained some peptide fragments exerting antimicrobial activity.