Abstract: To effectively control quality and shelf life of fermented fish, strains, which can be used as starter culture, were separated and screened from traditional fermented Trachinotus ovatus. Using catalase positive, salt tolerance, antibacterial properties and histamine-free as the valuation indexes, the target strains were isolated by dilution separation method, and strains biological characteristics, flavor and tastes of metabolites were evaluated. The experimental results showed that two potential fermented strains (zh-b and zh-f) were screened from 38 strains, which were isolated from the fermented Trachinotus ovatus. According to molecular biological identification, two strains were Pediococcus pentosaceus and Lactobacillus plantaruma, and their most appropriate temperatures were 20 and 30 ℃, the optimal pH was 5 and 6, respectively. Both strains had antibacterial activity against Staphylococcus aureus and Escherichia coli, but the Lactobacillus plantarum showed the better antibacterial activity, and the acid production capacity was far higher than that of Pediococcus pentosaceus. Fermentation products were determined by electronic nose, it was and found that methyl groups, sulfides, nitrogen oxides, alcohols or aldehydes and ketones and organic aromatic sulfides produced by Pediococcus pentosaceus (64.10, 57.98, 44.75, 40.22 and 19.93) were higher than those of Lactobacillus plantarum (5.50, 34.11, 10.68, 0.00 and 8.95), which was a major contributor to aroma components. The electronic tongue test showed that both strains had the ability to produce umami and fresh aftertaste, and the taste values of Pediococcus pentosaceus were 14.32 and 6.64, which were significantly higher than those of Lactobacillus plantarum, which produced umami and fresh aftertaste (3.20 and 2.90), but only Lactobacillus plantarum produced a sour taste. In addition, both strains also produced a very small amount of bitterness. The research would provide a basis for the large-scale production of fermented Trachinotus ovatus using the two strains as a mixed starter.