Abstract: Objective: In order to investigate the use of by-products from aquatic product processing in the preparation of antibacterial hydrolysates and their antibacterial effects, in this study, the specific spoilage organisms of Penaeus vannamei (PV-SSOs) were used as the experimental bacteria and the antibacterial hydrolysates from shrimp processing by-products (SPPH) were prepared, as well as the antibacterial effects of SPPH on PV-SSOs were determined. Methods: Five different proteases were used to hydrolyze the processing by-products of Solenocera crassicornis, and the antibacterial effects of these hydrolysates generated on PV-SSOs were compared. An appropriate protease was selected to prepare SPPH. The effects of hydrolysis conditions including enzyme addition content, hydrolysis time, hydrolysis temperature and solid-liquid ratio (w/v) on the inhibition of PV-SSOs were determined. Then, the response surface methodology was applied to optimize the hydrolysis conditions for SPPH preparation. Furthermore, the molecular weight distribution of peptidic fractions in SPPH was analyzed using high-performance liquid chromatography (HPLC). In addition, the effect of SPPH on the cell membrane permeability of PV-SSOs was measured using membrane leakage method, and the microstructure changes of PV-SSOs after SPPH treatment were further observed through scanning electron microscopy (SEM). Results: Pepsin was selected as the appropriate protease. Under the conditions of pH2.0 and solid-liquid ratio 1:2 (w/v) in the hydrolysis reaction, the optimal conditions for antibacterial hydrolysates preparation that inhibited PV-SSOs were obtained through the Box-Behnke experiment using response surface methodology at three levels and three factors as follows: 700 U/g of pepsin addition, 2.3 h of hydrolysis time, and 33 ℃ of hydrolysis temperature. The diameter of the inhibitory zone of SPPH on PV-SSOs was 24.10±0.43 mm. The result of HPLC showed that the relative percentage of peptidic fractions in SPPH with molecular weight less than 3000 Da was close to 70%. After treated with SPPH for 2 h to 12 h, the cell membrane permeability of PV-SSOs was significantly increased as compared to the control of PV-SSOs (P<0.05). Under SEM observation, some bacteria of PV-SSOs were twisted and shrunk, forming depressions, pores, and exudation of contents on the surface of the cell membrane after SPPH treatment for 12 hours. Conclusions: The processing by-products of Solenocera crassicornis can be applied to prepare antibacterial hydrolysates. SPPH inhibits PV-SSOs through membrane damage mode. All these findings will provide a theoretical basis for further development of SPPH for the preservation of Penaeus vannamei.