Aim To date, there is a controversy regarding the effects of acute aerobic exercise of moderate intensity on executive function and prefrontal cortex activity in community-dwelling older adults. This study aimed to investigate the effects of acute aerobic exercise of moderate intensity in healthy older adults. Methods Fifty-six healthy older adults were randomly assigned to the experimental group (EG) that performed moderate-intensity aerobic exercise or the wait-list control group (CG) for a total of 15 sessions. To compare the two groups, the Stroop Color-Word Task (SCWT) and the Trail-Making Test Part B (TMT-B) were implemented for executive function and prefrontal cortex activity during executive function testing. Results After the intervention, the EG achieved a significant improvement in the SCWT (P textless 0.001; η2 = 0.196) and the TMT-B (P textless 0.001; η2 = 0.245) compared with the CG. In addition, the EG showed a lower HbO2 concentration in the prefrontal cortex than the CG during the SCWT (P textless 0.05; η2 = 0.076) and the TMT-B (P textless 0.05; η2 = 0.090). Conclusions These findings shed new light on the clinical effectiveness of acute moderate-intensity aerobic exercise on executive function and prefrontal cortex activity in healthy older adults.