Previous studies have indicated that changes in oxygenated hemoglobin concentration (O₂Hb) in the prefrontal cortex (PFC) are associated with changes in cognitive function. Therefore, the present study aimed to explore the effect of differences in O₂Hb levels in the left and right PFC (L-PFC and R-PFC, respectively) on cognitive function after exercise. This study included 12 healthy male college students. The exercise regimen consisted of 4 min of warm-up and rest each, followed by 20 min of moderate-intensity exercise and 20 min of post-exercise rest. Participants underwent the 2-back cognitive test thrice (pre-exercise, post-exercise, and after the 20 min post-exercise rest period), and their reaction times were recorded. O₂Hb levels in the PFC were monitored using functional near-infrared spectroscopy. We analyzed the correlations between changes in post-exercise reaction times and differences in peak O₂Hb levels (L-PFC minus R-PFC), area under the curve for O₂Hb changes, and increases in the O₂Hb slope during exercise. Peak O₂Hb, area under the curve (AUC) for O₂Hb change, and increase in the slope of O₂Hb were significantly correlated with changes in reaction time. These findings provide insight into the mechanism by which O₂Hb differences between the L-PFC and R-PFC affect cognitive function.