Quorum sensing in spoilage bacteria of aquatic products is one of the reasons to accelerate the spoilage of aquatic products. In recent years， due to the use of chemical preservatives， many spoilage bacteria have developed resistance， and their safety also worries consumers， so the research on safe and efficient biological preservatives is an important topic in aquatic preservation. In this study， we tried to use immobilized penicillin acylase to inhibit the quorum sensing of Pseudomonas fluorescens. The quenching effect of immobilized penicillin acylase on Pseudomonas fluorescens AHLs was verified by CV026； the effect of enzyme on biofilm was studied by optical microscope and scanning electron microscope； the activity of extracellular protease was verified by milk agar plate； the interaction between enzyme and different AHLs was predicted by molecular docking. The results showed that the immobilized penicillin acylase could reduce the content of AHLs in Pseudomonas fluorescens， thus reducing the production of purpurin in Pseudomonas fluorescens CV026 and the formation of Pseudomonas fluorescens biofilm and extracellular polymer. At the same time， it also interfered with the expression of extracellular protease. When the enzyme mass concentration was 15 mg/mL， no obvious protein inhibition circle could be observed. Molecular docking results showed that the enzyme could successfully dock with both short chain and long chain signaling molecules， but preferred to long chain AHLs. Compared with C4-HSL， C14-HSL was completely wrapped by amino acid residues around the binding capsule and connected with each other by hydrogen bonds， with more hydrogen bonds and stronger binding. It can be concluded that the immobilized penicillin acylase has good quorum sensing quenching activity and can be used as a new preservative for aquatic products.