African swine fever (ASF) is a highly fatal viral disease that poses a severe threat to the global swine industry. Developing a safe and effective vaccine remains an urgent and complex challenge. Lactobacillus plantarum (L. plantarum), recognized as a food-grade antigen delivery vehicle, has demonstrated great promise in vaccine development and application for various infectious diseases. To overcome the weak intestinal immunogenicity and short mucosal residence of conventional vaccines, we employed the antibiotic-free selection system of L. plantarum NC8. By fusing the nontoxic, intestinal-epithelium-targeting cholera toxin B subunit (CTB) with key ASFV antigens (p30, p54, p49, p22, pK205R, and pE248R), we constructed five newly engineered lactic acid bacteria (LAB) strains which were subsequently administered to mice orally, either individually or as a multivalent cocktail. Experimental findings revealed that oral administration of the newly engineered LAB vaccine significantly promoted the activation of DCs within the intestinal mucosa. Moreover, this oral vaccine effectively triggered T-cell immune responses in both the mucosal and systemic immune compartments and markedly enhanced B-cell activation in the intestinal mucosa. Further immunological analyses confirmed that the vaccine significantly elevated serum antigen-specific IgG and intestinal antigen-specific IgA levels. Collectively, these results indicate that the cocktail of newly engineered LAB strains elicits potent humoral, cellular, and mucosal immune responses against ASFV in mice. This novel oral immunization strategy not only provides a strong theoretical foundation for developing an oral ASFV vaccine but also contributes valuable insights for advancing oral vaccines capable of inducing systemic immune protection through mucosal immunity.IMPORTANCEAfrican swine fever virus (ASFV) is a highly virulent pathogen that continues to inflict severe economic losses on the global swine industry. In this study, we developed an environmentally friendly oral vaccine candidate utilizing an antibiotic-free selection system based on L. plantarum. A cocktail of newly engineered LAB strains, each expressing distinct ASFV antigenic proteins (p30, p54, p49, p22, pK205R, and pE248R), was administered to mice via oral gavage to evaluate its protective potential against ASFV infection. The oral delivery of this newly engineered LAB cocktail elicited robust humoral, cellular, and mucosal immune responses, demonstrating significant enhancement of the host's immunological defense mechanisms against ASFV. Overall, this strategy presents a promising approach for the prevention of ASFV infection while effectively eliminating the risks associated with antibiotic residues, thereby offering a safer and more sustainable vaccination platform for the swine industry.