【Aim】This study investigated the antibacterial mechanism of the extract from a biocontrol bacterium isolated from tobacco rhizosphere soil against Ralstonia solanacearum, the causal agent of tobacco bacterial wilt, aiming to provide a new candidate strain for the biocontrol of this disease.【Method】Antagonistic bacteria with significant inhibitory effects against R. solanacearum were isolated from the rhizosphere soil of healthy tobacco plants via the plate confrontation assay. The selected strain was identified through morphological, physiological, biochemical, and molecular biological analyses. Its disease control and plant growth-promoting efficacy were evaluated through greenhouse experiments, and its colonization in tobacco rhizosphere soil and tobacco plants was detected via an antibiotic marker method. Furthermore, a 96-well plate method was employed to determine the minimum inhibitory concentration (MIC) of the bacterial extract against R. solanacearum, and thus evaluating the effect of the strain on R. solanacearum.【Result】A biocontrol bacterial strain, designated FY3-17, which exhibited significant inhibitory activity against R. solanacearum, was isolated from tobacco rhizosphere soil and identified as Serratia marcescens. Greenhouse experiment results showed that the fermentation broth of FY3-17 provided the control efficacy of 73.98% against tobacco bacterial wilt and significantly promoted tobacco plant growth. Furthermore, this strain stably colonized both the tobacco rhizosphere soil and the tobacco plants. On day 42 post-inoculation, the maximum colonization density (2.0×10⁴ CFU·g^-1) was observed in rhizosphere soil, followed by roots (5.35×10³ CFU·g^-1), stems (8.90×10² CFU·g^-1), and leaves (4.57×10¹ CFU·g^-1). Additionally, the extract of FY3-17 significantly inhibited the growth of R. solanacearum, with an MIC of 40 μg·mL^-1, and it interfered with the biofilm formation and extracellular polysaccharide production.【Conclusion】The S. marcescens strain FY3-17 screened in this study demonstrates considerable potential for controlling tobacco bacterial wilt and promoting plant growth, indicating promising prospects for development and application.