Vibrio alginolyticusis the common pathogen affecting various species of marine organisms. It has been demonstrated thatfliRis a necessary virulence factor to adhere and infect their hosts for pathogenic bacteria. Frequent disease outbreaks in aquaculture have highlighted the necessity of developing effective vaccines. In the present study, in order to investigate the function offliRinV.alginolyticus, thefliRdeletion mutant ΔfliRwas constructed and its biological properties were evaluated, additionally, the differences in gene expression levels between wild-type and ΔfliRwere analyzed by transcriptomics. Finally, ΔfliRwas used as a live attenuated vaccine to immunize grouper via the intraperitoneal route to evaluate its protective effect. Results show thatfliRgene ofV. alginolyticuswas identified as being 783 bp in length, encoding 260 amino acids, and showing significant similarity to homologs of otherVibriospecies. ThefliR-deletion mutant ΔfliRofV. alginolyticuswas successfully constructed, and its biological phenotype analysis showed no significant differences in growth capacity and extracellular enzyme activity compared to the wild-type. However, a substantial reduction of motility ability was detected in ΔfliR. Transcriptomic analysis revealed that the absence offliRgene is responsible for a significantly decreased expression of flagellar genes, includingflaA,flaB,fliS,flhBandfliM. ThefliR-deletion mainly affects the related pathways involved in cell motility, membrane transport, signal transduction, carbohydrate metabolism, and amino acid metabolism inV. alginolyticus. The efficacy of ΔfliRas a candidate of live attenuated vaccine were evaluated by intraperitoneal injection in grouper. The ΔfliRprovided the RPS (Relative protection rate) of 67.2% againstV. alginolyticusin groupers. The ΔfliRefficiently stimulated antibody production with specific IgM still detected at 42 d post-vaccination, and significantly elevated the activity of antioxidant enzymes like Catalase (CAT), Superoxide dismutase (SOD), and lactate dehydrogenase (LDH) in the serum. The higher expression levels of immune-related genes were observed in the immune tissues of inoculated grouper compared to the control. In conclusion, ΔfliReffectively improved the immunity of inoculated fish. The results suggest that ΔfliRis an effective live attenuated vaccine against vibriosis in in grouper.