Objective To evaluate the performance of rural practitioners of endoscopic cleaning and disinfection participating in the Early Diagnosis and Treatment Program for Upper Gastrointestinal Cancers and to analyze the influencing factors. Methods The questionnaires for skill assessment were designed based on the skill scheme and clinical practice of the Early Diagnosis and Treatment Program for Upper Gastrointestinal Cancers in rural China, and the App Early diagnosis, Early treatment was used as the examination platform. The practitioners in 539 county hospitals from 25 provinces participating in the program in 2019 were assessed for techniques and skills for endoscopic cleaning and disinfection and the excellence rate was calculated. Multivariate logistic regression model was used to analyze the influencing factors for the examination. Results A total of 1 671 endoscopic cleaning and disinfection practitioners participated in the assessment with the score of 73.41±16.60. The passing rate was 85.82%, and the excellence rate was 44.94%. Among all questions, the correct rate of "opportunistic screening flow chart" was the highest (98.21%), and that of "the evaluation index for mass screening" was the lowest (57.89%). The multivariate logistic regression analysis showed that the excellence rate was high in practitioners who had a bachelor degree or above (OR=1.627,95%CI:1.319-2.007,P<0.001), the career for 5 to <15 years (5 to <10 years:OR=1.329,95%CI:1.045-1.689,P=0.020; 10 to <15 years:OR=1.384,95%CI:1.026-1.867,P=0.033), working in eastern and central regions (eastern regions: OR=3.476,95%CI:2.368-5.103,P<0.001;central regions: OR=4.028,95%CI:2.679-6.057,P<0.001) and with full understanding of the screening scheme (OR=1.547,95%CI:1.246-1.921,P<0.001) . Conclusion Practitioners on the Early Diagnosis and Treatment Program for Upper Gastrointestinal Cancers in rural China have mastered the basic screening scheme and skills for endoscopic cleaning and disinfection. The education background, duration of the career, area and understanding of screening scheme are influencing factors for the excellence rate of endoscopic cleaning and disinfection.