Pseudomonas fluorescens was dominant spoilage bacteria in cultured fishes during the chilled storage. In the present study， the biofilm formation and spoilage phenotypes of five P. fluorescens isolates were comparatively characterized. The biofilm forming ability and adhesion of the isolates were measured by using crystal violet staining， phenol sulfuric acid assay， bead vortexing method and fluorescence microscopy. Various spoilage phenotypes inculding swimming motility， protease activity， siderophore were performed. The results showed that five P. fluorescens isolates grew fast in LB media at 28 ℃， and formed thick pellicle at the air liquid（A-L） interface for 24 h. All of them produced rapidly biofilm on microtiter plates， and PF01, PF06, PF07 and PF10 reach the maxmium amount of biomass at 12 h， however， PFuk4 arrived at 18 h. The isolates produced continuously exopolysaccharides with the extension of time and arrived to the maxmium amount at 18-24 h. Five isolates adhered to stainless steel quickly， possessed high swimming motility and protease activity， as well as production of siderophore. Only PF07 and PFuk4 were detected to short chain AHLs activity， which could be associated with higher ability of biofilm formation， adhesion， swimming and protease. The present study provided a good fundation to clarify the spoilage mechnism of P. fluorescens based on AHLs.