Fish serum may reflect status of many biochemical processes in the metabolism. Heavy metals, as environmental stressors, may alter serum biochemical parameters in fishes. Thus, freshwater fish, Oreochromis niloticus, were exposed to low levels (0.05 mg/L) of metals (silver [Ag], cadmium [Cd], copper [Cu], chromium [Cr], zinc [Zn]) to investigate responses of serum biochemical parameters over different exposure periods (0, 5, 10, 20, 30 d). Fish mortality occurred only in Ag exposure, as all fish died between days 12 to 16. Activities of alkaline phosphatase (ALP), alanine transaminase (ALT), and aspartate transaminase (AST) were altered only in Cu- and Cd-exposed fish. Both Cd and Cu exposures decreased the activity of ALP, although they increased the activities of ALT and AST. Glucose concentrations increased in Ag-, Cd-, and Cu-exposed fish, with a sharp increase occurring in Ag-exposed fish before mortality began. Total protein and triglyceride concentrations increased in Ag-exposed fish, although they decreased in Cu-exposed ones. However, all metal exposures increased cholesterol concentration in the serum. Concentration of blood urea nitrogen increased in Ag-, Cd-, and Cu-exposed fish, although it decreased in Cr-exposed ones. Calcium level decreased only in Cu-exposed fish, and Cl− level decreased in Ag-exposed fish. Silver and Cu exposures also decreased Na+ level in the serum. Cadmium and Cu exposures increased serum K+ levels. The present study, investigating the effects of environmentally realistic metal exposures on serum biochemical parameters, demonstrated that fish serum could sensitively reflect environmental metal stress. Thus, it suggests that serum biochemical parameters could be used as important and sensitive biomarkers in ecotoxicological studies concerning the effects of metal contamination and fish health.