[Background] Bacterial ghosts (BGs) are empty and intact bacterial envelopes of Gram-negative bacteria that are produced by controlled expression of the phage PhiX174 lysisE gene. BGs have higher biological safety, which can induce good systemic and mucosal immune response in a similar way of live bacteria. [Objective] To identify the pathogenic strain 16-3 isolated from the liver of large yellow croaker (Pseudosciaena crocea) with ulcerative disease and to generate bacterial ghosts from this strain by the temperature-controlled expression of cloned bacteriophage PhiXl74 lysis gene E. This study will provide an effective means to prevent and control the infection of Vibrio alginolyticus in aquaculture fish species. [Methods] Strain 16-3 was identified by morphological, physiological and biochemical characteristics and 16S rRNA gene sequence analysis. Temperature-controlled lysis plasmid pBV220-LysisE was constructed and then electroporated into V. alginolyticus strain 16-3 to form recombinant strain 16-3(pBV220-LysisE). Different initial concentration cultures of recombinant strain 16-3(pBV220-LysisE) were induced at 42 °C to compare the differences of lysis kinetics curves and lysis rate among them. V. alginolyticus strain 16-3 ghosts were generated under the optimum condition, and then their morphology and structure were observed by electronic microscope, and the viable cell counts in the lyophilized ghosts were measured using a pour plate method. [Results] The pathogenic strain 16-3 was identified as V. alginolyticus according to morphological features, physiological and biochemical characteristics and 16S rRNA gene phylogenetic analysis. The temperature-controlled lysis plasmid pBV220-LysisE and recombinant V. alginolyticus strain 16-3(pBV220-LysisE) were constructed, respectively. The optimum conditions for V. alginolyticus strain 16-3 ghosts generation were as follows: the induction was carried out by selecting the initial concentration of cultures reached an OD600 of 0.3. The bacterial ghosts could be harvested after induced for 3 h and the lysis rate of strain 16-3 ghosts was 96.9%. However, there was no residual bacteria found on plates with lyophilized 16-3 ghosts inoculation. Electron micrographs clearly showed that no gross alterations in cellular morphology compared to unlysed cells except for the obvious lysis pore on the cell surface and cell shrinkage due to the loss of cytoplasmic materials. [Conclusion] V. alginolyticus strain 16-3 ghosts were generated in this study, which provided a basis for the further use as a vaccine or vaccine delivery vector.