Environmental and unfavourable circumstances significantly impact a fish's serum and blood biochemical parameters. The study examines the impact of long-term exposure to salinity stress on the blood and immune parameters of rainbow trout (Oncorhynchus mykiss) juveniles weighing 80 grams in farms with three different salinity levels in Golestan province (Iran): 0.5 ppt (freshwater), 3 ppt (brackish water), and 18 ppt (saltwater). After being purchased and transferred to the farms, the juveniles were fed a commercially available salmon diet for five months. After the trial period ended, blood samples were obtained from 60 healthy fish, with each fish weighing 250 grams at each farm. The samples were subjected to centrifugation in order to separate the serum from the blood cells. The desired parameters were measured using conventional laboratory techniques. The results revealed significant differences in the levels of white blood cells (WBCs) and immunoglobulin M (IgM) among the three experimental groups (p < 0.05). The fish in seawater showed the highest white blood cell count (14300 ± 310 mm3) and immunoglobulin M (0.428 ± 0.007 g/l) values. Red blood cell counts (RBCs), haematocrit (HCT), haemoglobin (Hgb), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), complement C3 and complement C4 were all significantly lower in fish raised in brackish water compared to fish raised in freshwater and saltwater (p < 0.05). These parameters did not show any significant differences between fish reared in freshwater and saltwater (p > 0.05). The levels of total protein, glucose, cortisol, albumin, cholesterol, triglyceride, urea, uric acid, creatinine, alkaline phosphatase, lipase, and amylase were significantly higher in fish raised in saltwater compared to fish raised in fresh and brackish waters (p < 0.05). Significantly elevated levels of calcium, phosphorus, chloride, iron, copper, zinc, and magnesium ions were detected in saltwater fish that were raised (p < 0.05). Simultaneously, the amounts of sodium and potassium exhibited no statistically significant variations across all treatments (p > 0.05). To summarize, this research emphasizes that rainbow trout, a euryhaline species, exhibit better growth and survival rates when reared in water with a salinity of 18 parts per thousand (ppt). Furthermore, this species exhibits enhanced resistance to stressors at this specific salinity level, resulting in a more favourable habitat since these indicators tend to return to their original values observed in freshwater.