Objective To study the characterization of surface electromyographic signals of upper limb force-generating muscles operated by rolling manipulation of Tuina doctors. Methods Surface electromyographic signals during rolling manipulation were collected from beginners and proficient operators for comparative analysis, and the patterns, similarities, and differences were summarized. Results The iEMG ratio of the ulnar lateral wrist extensor muscle in the proficient group was significantly higher than that in the beginner group (P<0.05), while that of the middle deltoid fascicle in the beginner group was significantly higher than that in the proficient group (P<0.05). There was no statistical difference in the iEMG ratios of other muscle groups (P>0.05). The proficient group mainly used the ulnar wrist extensors, lateral head of triceps brachii, pectoralis major, and ulnar wrist flexors, while the beginner group mainly used the lateral head of triceps brachii, pectoralis major, and radial wrist flexors. Conclusions The proficient group and the beginner group shared common features in the electromyographic signals of the rolling manipulation. The proficient group increased the angle of ulnar deviation by recruiting more ulnar wrist extensors to increase the angle of palmar flexion and the dorsal contact area of the hand, and the proficient group used the middle and posterior deltoid fasciculus muscles less intensively and mastered the ‘sinking shoulder’ principle better.