The research investigated the biochemical properties of spoilage bacteria Pseudomonas fragi (P. fragi) in grouper (Epinephelus lanceolatus) during cold storage. The proteolytic activity and the spoilage-related metabolic pathway were characterized by whole genome sequencing. The results revealed optimal protease activity at pH 7.0 and 40 °C, with significant inhibition observed in the presence of protease inhibitors, organic solvents, and metal ions. The kinetic analysis demonstrated strong substrate affinity, while the thermodynamic parameters indicated excellent thermal stability. Freshness index evaluations confirmed the potential of fish protein degradation of P. fragi. The whole genome sequencing has made gene-level protease analysis a crucial approach for investigating protease metabolism. Genes associated with protein degradation and spoilage pathways were identified. The results revealed that both P. fragi V11 and SS strains contain 36 protease-encoding genes, with metalloprotease and serine protease representing the predominant proteases in P. fragi. These identified genes functioned in regulating amine, sulfur and amino acid metabolism. Whole genome sequencing may offer valuable insights into protease mechanisms and protein degradation processes.