Strenuous exercise can induce transient changes in serum enzyme activities, with the magnitude of these responses varying according to training and biomarker characteristics. This study compared immediate pre-to-post exercise changes in creatine kinase (CK), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) between trained basketball players and non-athletes following exhaustive incremental cycling. Twenty young men (10 basketball players and 10 non-athletes) completed an incremental cycling test to volitional exhaustion. Blood samples were collected immediately before and after exercise. Group × Time interactions were analyzed using mixed-design analysis of variance with Holm adjustment, and a baseline-adjusted ANCOVA was performed for AST. Significant Group × Time interactions were observed for CK, LDH, and AST, but not ALT. CK increased by 5.6 ± 4.35 U/L in basketball players and 53.2 ± 20.06 U/L in non-athletes (between-group difference in change, −47.60 U/L; 95% CI, −62.09 to −33.11; Hedges’ g = −3.14). LDH increased by 8.2 ± 8.09 and 35.2 ± 26.60 U/L, respectively (between-group difference in change, −27.00 U/L; 95% CI, −46.43 to −7.57; Hedges’ g = −1.32). AST increased by 1.1 ± 1.66 and 2.9 ± 1.60 U/L, respectively, and the group effect remained significant after baseline adjustment, although pre-exercise AST was higher in basketball players. No differential immediate ALT response was observed. Thus, immediate serum enzyme responses differed by training status and biomarker. Given differences in exercise duration and maximal workload between groups, these findings should be interpreted as responses to individually maximal exercise rather than an identical exercise dose.