To study the physiological performance of Nile tilapia (Oreochromis niloticus) under long term nitrite exposure stress, five different treatments of nitrite concentration groups (control, 0.03, 0.06, 0.12, and 0.23 mmol/L) were set to assess the effects of the physiological, biochemical, metabolic function, and non-specific immune response in blood and liver tissue of juvenile tilapia under 21 days of nitrite exposure treatments. The results showed that the white blood cell count in the blood increased slightly with the increasing nitrite nitrogen concentrations, whereas the red blood cell count and hemoglobin concentration decreased significantly (P<0.05). Serum glucose concentration, alanine aminotransferase, aspartate aminotransferase activities, and K⁺ concentration in juvenile tilapia increased significantly with increasing nitrite nitrogen concentrations (P<0.05), whereas triglycerides, total cholesterol, total protein, Na⁺, and Cl^- concentration, and superoxide dismutase, alkaline phosphatase, and lysozyme activities, and immunoglobulin concentrations showed an inverse trend with increased nitrite nitrogen (P<0.05). The hepatic expression of non-specific immune factors HSP70, Il-1β, IL6, and TNFα mRNA levels was significantly improved by increased nitrite nitrogen concentrations (P<0.05), whereas LYS mRNA expression decreased. The results suggested that long-term exposure of low nitrite nitrogen concentrations (0.06-0.23 mmol/L) could lead to dysfunction of blood physiology, metabolism, and immune response in tilapia, and furthermore the hepatic non-specific immune gene expression could be affected. Data from this study will be useful to further understand nitrite ion toxicity mechanisms in the tilapia, and thereby help to establish a safe range of nitrite nitrogen in tilapia aquaculture.