Qigong is a gentle exercise that promotes health and well-being. This study evaluated the acute physiological and psychological effects of one session of qigong exercise in older practitioners. A total of 45 participants (mean age, 65.14 years) were recruited. Meridian electrical conductance, State-Trait Anxiety Inventory (STAI), heart rate variability (HRV), and Short Form 36 (SF-36) were evaluated and compared before and after one session of qigong exercise. The results revealed that the electrical conductance of all meridians, except spleen and bladder meridians, increased significantly (p < 0.05). Compared with baseline values, upper to lower body ratio and sympathetic/vagal index were significantly improved and closer to 1 (p = 0.011 and p = 0.007, resp.). STAI-S and STAI-T scores decreased significantly (p < 0.001 and p = 0.001, resp.). The RR interval of HRV decreased significantly (p = 0.035), a significant positive correlation was observed between kidney meridian electrical conductance and SF-36 physical scores (r = 0.74, p = 0.018), and a positive correlation was observed between pericardium meridian electrical conductance and SF-36 mental scores (r = 0.50, p = 0.06). In conclusion, one session of qigong exercise increased meridian electrical conductance, reduced anxiety, and improved body and autonomic nervous system balance. These findings provide scientific evidence for acute physiological and psychological effects of qigong exercise in older practitioners.