Saline-alkaline Oreochromis niloticus Nile tilapia aquaculture is vital for expanding aquatic production, but gill rot disease causes severe losses. Conventional antibiotic use breeds resistance, pollution, and safety risks, urging eco-friendly alternatives. This study first isolated and identified the pathogen responsible for gill rot in saline-alkaline cultured tilapia as Aeromonas veronii (strain ONG-1) through morphological, biochemical, and 16S rRNA phylogenetic analyses. It then evaluated the efficacy of astragalus polysaccharide (APS), chitosan (CS), and their combination (astragalus polysaccharides + chitosan, APS + CS) against this pathogen: in vitro tests revealed both CS (stronger) and APS exerted direct bactericidal activity. In vivo experiments demonstrated that APS + CS significantly reduced cumulative mortality by 38.1 % (from 70.0 % to 43.3 %), lowered bacterial loads in tissues (gill, liver, and head kidney) and blood, alleviated tissue edema, regulated hematological indices (balancing immune cell proportions), enhanced serum innate immunity (complement C3, lysozyme), improved oxidative balance (T-SOD, MDA), and modulated immune-related genes (pro-inflammatory cytokine IL-1β, anti-inflammatory cytokine TGF-β, and antimicrobial peptide hepcidin) by downregulating IL-1β, upregulating TGF-β and hepcidin (changing 2.3, 1.8, and 2.1 -fold in head kidney, respectively). The results showed that CS had strong early bactericidal activity (disrupting bacterial membranes), while APS had long-term immunoregulatory potential (enhancing antioxidant/cytokine responses); APS + CS synergized by combining immediate pathogen clearance and sustained immune enhancement. These findings clarify the pathogenic basis of tilapia gill rot in saline-alkaline water and validate APS + CS as a promising antibiotic alternative, providing theoretical support and technical guidance for sustainable disease control in such aquaculture systems.