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Background With the aging of the population, the prevalence of atrial fibrillation is increasing year by year. As the "gatekeepers" of community residents' health, grassroots medical staff with a high level of knowledge about atrial fibrillation is particularly important for standardized management of atrial fibrillation. Objective To analyze the mastery of atrial fibrillation-related knowledge among grassroots medical staff in Fengxian District, Shanghai and its influencing factors, thus, providing a theoretical basis for subsequent training work. Methods This study is a cross-sectional survey study. From June to July 2021, a systematic sampling method was used to select 1 393 grassroots medical staff as the research objects including general practitioners, nurses, pharmacists, public health physicians and other medical staff (imaging technology/physician, laboratory technician, traditional Chinese medicine doctor and so on) from 21 community health service centers in Fengxian District, Shanghai. This study used a self-developed electronic questionnaire to investigate them. The contents of the questionnaire included general information and knowledge related to atrial fibrillation (basic knowledge about atrial fibrillation, knowledge about anticoagulant therapy and knowledge about rhythm/heart rate control) . This study compared the mastery of atrial fibrillation-related knowledge among grassroots medical staff in different positions, and used univariate Logistic regression, ordinal multiple classification or binary Logistic regression to analyze the influencing factors of grassroots medical staff's mastery of atrial fibrillation-related knowledge. Results A total of 1 383 valid questionnaires were recovered, and the valid questionnaire recovery rate was 99.28%. Among 1 383 grassroots medical staff, 506 cases (36.59%) were general practitioners. 54.88% (759/1 383) , 97.69% (1 351/1 383) and 69.63% (963/1 383) failed in the basic knowledge about atrial fibrillation, knowledge about anticoagulation and knowledge about heart rhythm/heart rate control, respectively. There were statistically significant differences in the scores of atrial fibrillation knowledge, anticoagulation knowledge and heart rhythm/heart rate control knowledge among grassroots medical staff in different positions (P