Visceral white nodules disease (VWND), caused by Pseudomonas plecoglossicida, poses a significant threat to the large yellow croaker (Larimichthys crocea) aquaculture industry. Developing live attenuated vaccines (LAVs) requires a fine balance between safety and immunogenicity, which remains a primary challenge. In this study, we developed a stable and effective LAV by constructing a markerless triple-gene deletion mutant ΔmlaEΔompRΔdeoR of P. plecoglossicida. The mutant exhibited high genetic stability and maintained growth kinetics compared to the wild-type (WT) strain under host-relevant conditions. Vaccination trials demonstrated that the triple mutant was significantly attenuated and safe for L. crocea. Following WT challenge, the triple mutant provided a relative percent survival (RPS) of 80.43% via intraperitoneal (i.p.) injection and 76.92% via immersion. For i.p.-vaccinated group, a significant reduction in splenic bacterial colonization and the absence of macroscopic white nodules were observed post-challenge. Furthermore, i.p.-vaccination triggered a coordinated immune response, characterized by the early upregulation of pro-inflammatory cytokines followed by the sustained activation of adaptive immune-related genes and elevated serum-specific IgM levels. These findings demonstrate that the ΔmlaEΔompRΔdeoR mutant is a promising LAV candidate, providing a viable strategy for the integrated control of P. plecoglossicida infection.