To characterize the involvement of microorganisms in amino acid degradation and fish quality deterioration, three major grass carp spoilage bacteria were artificially inoculated in amino acid solutions (in-vitro) and grass carp flesh (in-situ). Results showed that Pseudomonas putida largely degraded free amino acids and produced 3.78 mM/100 g ammonia in grass carp flesh, relying on its high amino acid deamination1 activity. Aeromonas rivipollensis produced 3-Methyl-butanol and 2-Methyl-butanol through leucine and isoleucine degradation. Shewanella putrefaciens had potent ornithine-decarboxylation activity (423.91 × 10-9 µg/CFU) and released 22.98 mg/kg putrescine in situ. S. putrefaciens could produce more putrescine when cooperating with P. putida through the arginine deiminase pathway. To conclude, the biochemical activities identified through in-vitro tests correlated well with quality changes in inoculated grass carp flesh. The outcomes of this study provided fundamental information on the spoilage mechanisms of freshwater fish and important guidance for the development of quality control strategies.