Objective A retrospective cohort study was conducted to analyze the application value of Python programming in general education and comprehensive quality improvement of medical students. Methods A retrospective analysis was made on the application value of Python programming in the general education classroom of medical students from September 2020 to July 2021 by undergraduate students majoring in anesthesia in grade 2020, imaging in grade 2019, clinical in grade 2020, and laboratory sciences in grade 2020 in our university. A hundred students who used Python programming in general education class were divided into study group and control group. The teaching satisfaction, medical knowledge and lifelong learning ability, clinical skills, medical service ability, disease prevention, health promotion ability, interpersonal communication ability, and information management and research ability were compared between the two groups. Results In a comparison of teaching satisfaction between the two groups, the study group was very satisfied in 89 cases, satisfactory in 10 cases, and general in 1 case, and the satisfaction rate was 100.00%; the control group was very satisfied in 54 cases, satisfactory in 23 cases, general in 13 cases, and dissatisfied in 10 cases, and the satisfaction rate was 90.00%. The teaching satisfaction in the study group was higher than that in the control group, and the difference was statistically significant (P < 0.05). Compared with the control group, medical knowledge ability (basic knowledge, general education, and professional knowledge) and lifelong learning ability (learning concept and professional learning attitude) in the research group were significantly higher than those in the research group (P < 0.05). The scores of clinical skills (medical history analysis, basic diagnosis, treatment techniques, and disease analysis) and medical service ability (first aid ability, comprehensive analysis ability, and disease analysis ability) in the study group were significantly higher than those in the control group (P < 0.05). In terms of the ability of disease prevention and health promotion, the scores of disease prevention (health guidance, health education, and self-care) and health promotion ability (cooperative participation in diagnosis and treatment, guidance of medical and health work, and rational use of health resources) in the study group were higher than those in the control group, and the difference was statistically significant (P < 0.05). In the comparison of interpersonal communication ability, the scores of listening, expression, understanding, trust, medical terminology, and communication ability in the study group were higher than those in the control group, and the difference was statistically significant (P < 0.05). Comparing information management with research ability, the scores of information management ability (searching information, screening information, and sorting information) and research ability (arrangement ability, planning ability, and execution ability) in the research group were higher than those in the control group, and the data difference was statistically significant (P < 0.05). Conclusion The application of the Python programming method in general education and comprehensive quality improvement of medical students can effectively improve medical students' teaching satisfaction and medical knowledge such as lifelong learning ability, clinical skills, medical service ability, disease prevention, health promotion ability, interpersonal communication ability, and information management and research ability, which has a positive impact on the improvement of comprehensive quality.