ABSTRACT This study aimed to provide baseline data for the serum biochemical parameters of Brazilian sport horses undergoing physical training for eventing to be used as a practical guide. A total of 139 blood samples were collected by vacuum venipuncture of the jugular vein from healthy horses in training. Tubes without anticoagulant were used for biochemical analysis. Means and standard deviations were determined for aspartate aminotransferase (AST) (306.27±84.66 U/L), creatine kinase (CK) (181.06±80.38 U/L), urea (29.51±5.82 mg/dL), creatinine (1.44±0.20 mg/dL), gamma glutamyl transferase (GGT) (14.58±6.55 U/L), calcium (13.22±0.59 mg/dL), and albumin (2.86±0.16 g/dL). The specific reference ranges were: AST, 197-454 U/L; CK, 116-290 U/L; urea, 22-42 mg/dL; creatinine, 1.2-1.8 mg/dL; GGT, 9-26 U/L; calcium, 12.2-14.2 mg/dL; and albumin, 2.6-3.1 g/dL. Beneficial effects of the training program on the horses were observed and these effects did not lead to any changes in the biochemical parameters studied. The environment may influence the serum biochemical parameters, particularly AST and CK, of Brazilian sport horses.