Abstract: Bacterial biofilm formation is closely regulated by quorum sensing (QS). This study screened peptides targeting the LuxR protein of the QS system in Pseudomonas psychrophila (P. psychrophila) isolated from refrigerated squid, and investigated their inhibitory effects on biofilm formation. Candidate peptides were selected from half-fin anchovy (Setipinna taty) protein hydrolysate and Pacific white shrimp (Penaeus vannamei) Pelle protein-derived peptides. Molecular docking was employed to comparatively evaluate the binding interactions of N-acyl homoserine lactone (AHL) signal molecules and candidate peptides with the LuxR protein of P. psychrophila. Peptides exhibiting superior binding affinity relative to AHLs were selected as target peptides, and a series of derivative peptides were rationally designed through site-directed amino acid substitution. Derivatives with optimal LuxR-binding capacity were identified as targeted peptides, and the biofilm inhibition efficacy of both target and targeted peptides was subsequently assessed. Results showed that five candidate peptides (AAVVFMLR, PGK, FWAKSQL, MANRVGF, and HLRVGW) were screened. Only PGK successfully docked with six LuxR proteins, exhibiting a binding energy of –447.26 kcal/mol with the LuxR protein SSOs_2029, markedly lower than that of AHL-type signal molecules. However, PGK failed to dock with SSOs_1080. Through site-directed amino acid substitution, targeted peptides KGG and RGG were yielded, which successfully docked with SSOs_1080 with binding energies lower than most endogenous signal molecules. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration of peptides PGK, KGG, and RGG against P. psychrophila were 4 and 8 mg/mL, respectively. At 1/2 MIC (non-inhibitory to bacterial growth), RGG significantly outperformed PGK and KGG in biofilm inhibition (P<0.05). Collectively, targeted peptide RGG competitively displaces AHL signal molecules from the LuxR protein of P. psychrophila, thereby attenuating biofilm formation. These findings provide a theoretical foundation for the rapid screening of peptide-based QS inhibitors against this psychrophilic spoilage bacterium.