Abstract: The antiseptic effects of ethanol extract from vine tea on cooked fish were studied using the clump counts of growing bacteria as evaluation indicator. The main spoilage bacteria from cooked fish were isolated by dilution plate method and identified by 16S rDNA sequencing technology, and the antibacterial activity of ethanol extract was determined by filtering paper method. Besides, the inhibition mechanism was further investigated through measuring the influences on growth curve, conductivity and biomacromolecules contents. Results showed that the ethanol extract from vine tea could completely inhibit bacterial growth in cooked fish for 15 days with the final concentration of 30 mg·mL^-1, and the two strains of bacteria were isolated and identified as Bacillus methylotrophicus T-10 and Alcaligenes faecalis T-9. The ethanol extract showed neutral-sensitive antibacterial activities on these two strains with the minimum inhibitory concentrations（MIC） of 16.0 mg·mL^-1 and 20.0 mg·mL^-1. Further studies proved that ethanol extract could destroy the integrity of bacterial cell membrane and causing the spilt of macromolecular substances, thereby exerting the inhibitory effects.