In this study, after 30 days of long-time exposure to high salinity (36‰ and 44‰), we explored the combined effects of high salinity and ammonia nitrogen (15 mg/L ammonia-N) stresses on the energy metabolism, immune response, oxidative resistance, and ammonia metabolism of Litopenaeus vannamei during the 48 h of ammonia exposure. The results showed increased glucose levels in the hemolymph and accelerated anaerobic metabolism with higher lactate levels in the muscle of all experimental groups. The combined action of the stressors decreased the non-specific immune responses of shrimp by decreasing the activities of alkaline phosphatase (AKP) and acid phosphatase (ACP), and the expression levels of phenol oxidase (PO), immune deficiency (IMD), and arginine kinase (AK) genes at the end of the exposure. In addition, the antioxidant defense system that L. vannamei employs via the total antioxidant capacity (T-AOC), superoxide dismutase (T-SOD) and catalase (CAT) enzymes, and the heat shock protein70 (HSP70) and thioredoxin (TRX) proteins was affected by exposure to high salinity and amminoa-N when exposed to ammonia-N with different high salinity stress. Furthermore, the expression levels of ammonia metabolism-related genes glutamate dehydrogenase (GDH) and glutamine synthetase (GS) were initially significantly upregulated and then declined in the hepatopancreas of the high salinity treatment groups. The study revealed that variations in high salinity and ammonia beyond the optimal range significantly influence glycometabolism, non-specific immunity, antioxidation, and ammonia metabolism, and that these two factors can have significant synergistic effects.