Pseudomonas spp. are specific spoilage bacteria of various fish from freshwater and marine during aerobic chilled storage. The behavior of psychrotrophic Pseudomonas is one of the common causes of quality deterioration in refrigerated fish products. P. fluorescens as a typical psychrotrophic spoilage bacterium, is main dominant spoilage Pseudomonas in aquatic product. Whole genome sequence analysis of genetic information is helpful to understand the molecular basis of spoilage, drug resistance and adaptation of foodborne spoilage bacteria. Currently, about 30 strains of P. fluorescens with complete genome map have been published in NCBI database, of which only an isolate P. fluorescens PF08 comes from Scophthalmus maximus. To explore the strong spoilage potential and environmental adaptability of P. fluorescens, the proteolytic activity and total volatile basic nitrogen (TVB-N) production of two isolates of P. fluorescens PF07 and PF08 from marine fish were measured and the mechanisms of spoilage and adaptation were explored deeply by comparative genomics. P. fluorescens PF07 and PF08 showed strong proteolytic activity and produced high TVB-N in refrigerated fish juice. After sequencing, assembly and functional annotation of whole genome, the length of the PF07 genomic was 6.13 Mb, and GC content was 61.4%. According to pan genome analysis, there were 4980 core genes in the two isolates, and 516 and 470 unique genes in PF07 and PF08, respectively. The highest proportion of genes was involved in amino acid metabolism both in the two isolates by COG and KEGG annotations, while the most unique genes belonged to inorganic ion transport and metabolism in PF07. Glycosyltransferases and glycoside hxydrolases genes accounted for the highest proportion among carbohydrate active enzyme in the two genomes based on CAZy annotation. In addition, many genes of enzymes and related protein associated with degrading various substrates of carbohydrates, proteins, and lipids were founded, such as alkaline metalloproteinase AprA, polyamine ABC transporter permease PotC, arginine and ornithine decarboxylase, etc. Several genes of sigma factors, including rpoS, rpoN and rpoD, were also distributed in the two strains. Two P. fluorescens isolators indicated strong spoilage potential, and had many genes encoding protease and putrescine formation and amino acid metabolism, and decomposing glycogen and fat, were distributed in the two isolates, as well as environmental adaptation regulators. This work preliminarily clarified the molecular basis of P. fluorescens with strong degradation of fish nutrition substrates at the genetic level, and revealed the metabolic characteristics and spoilage mechanism in P. fluorescens, which will provide a theoretical basis for exploring the spoilage mechanism of Pseudomonas in aquatic fish.