The intensification of aquaculture practices has led to frequent occurrence of diseases in farmed animals. While antibiotics and other chemicals are capable of disease control, prolonged misuse may result in adverse effects on the health of farmed animals. Thus, there is an urgent need to identify healthy and effective alternatives to antibiotics. This study aimed to assess the impact of fish-derived Lactobacillus acidophilus AC on the growth performance, liver antioxidant and immune capacity, intestinal morphology, intestinal digestive enzyme activity, intestinal flora, metabolism, and resistance to Aeromonas hydrophila of adult zebrafish. The experiment comprised of four groups: AC0 (the control), AC6 (106 CFU/mL L. acidophilus AC), AC7 (107 CFU/mL L. acidophilus AC), and AC8 (108 CFU/mL L. acidophilus AC). Each group had three replicates, and the experimental duration was 30 days. Results indicated that after supplementing with L. acidophilus AC, the growth performance, liver antioxidant capacity, and immune enzyme activity of adult zebrafish significantly increased. Intestinal health and digestive enzyme activity also showed marked improvement. Analysis of the intestinal flora revealed that L. acidophilus AC modulated the structure of intestinal flora, enhancing nutrient absorption and immunity by increasing the abundance of Bacteroidetes and Lachnospiraceae. Intestinal metabolomics analysis demonstrated that L. acidophilus AC significantly altered the intestinal metabolites, with 107 CFU/mL supplementation promoting the production of antioxidant and antimicrobial-related metabolites such as BetavulgarosideVIII and 7,10,13-hexadecatrienoic acid. Furthermore, supplementation with L. acidophilus AC enhanced the resistance to A. hydrophila of adult zebrafish. In conclusion, L. acidophilus AC effectively enhanced the health and immunity, with the addition of 8.87 × 106 CFU/mL and 8.89 × 106 CFU/mL L. acidophilus AC proving particularly beneficial for the growth and development of adult zebrafish.