Pseudomonas fluorescens is a widespread spoilage bacterium in food, and its spoilage characteristics and biofilm formation are regulated by N-acyl-homoserine lactone (AHLs) quorum sensing (QS) system. Quorum quenching (QQ) is considered as an effective strategy to control the spoilage bacteria. Therefore, this study revealed a new QQ bacteria Lactiplantibacillus plantarum YP4-1-2. Notably, the crude cell extract (CCE) from L. plantarum YP4-1-2 showed strongly QQ activity against the AHLs of P. fluorescens. The degradation rate of AHLs (N-octanoyl-L-homoserine lactone) reached 25.90%, 78.57% and 100% at the CCE protein concentrations of 57, 86 and 114 μg/mL, respectively. In addition, the CCE could drastically reduce the formation of biofilm, bacterial motility (swimming and swarming), and the release of extracellular protease and biogenic amines of P. fluorescens. Real-time polymerase chain reaction (PCR) results revealed that the CCE downregulated QS-related genes (rhlI, rhlR, aprX, algA, orm, flgA, and ldcA). Finally, using whole-genome sequencing analysis, the active substance in the CCE was identified to be penicillin V acylase (PVA) and named LpPVA. Meanwhile, homologous modeling and molecular docking analysis showed that the binding effect of LpPVA on long-chain AHLs was better than that on short-chain AHLs. This study demonstrated that L. plantarum YP4-1-2 could be considered as a promising QS inhibitor and antibiofilm agent against foodborne spoilage bacteria.