ObjectiveTo develop a questionnaire on the knowledge needs of respiratory rehabilitation training for in-service healthcare workers, and test its reliability and validity. MethodsA theoretical framework was constructed based on the contents of the respiratory rehabilitation course modules of American Association of Cardiopulmonary Rehabilitation and American Association for Respiratory Care. The first draft of the questionnaire was prepared through literature searches and analyses, semi-structured interviews, and expert group discussions. The reliability and validity of the questionnaire was tested by interviewing 17 respiratory rehabilitation professionals and 16 respiratory rehabilitation experts by correspondence, and investigating 380 cases of in-service healthcare workers engaged in respiratory rehabilitation. ResultsThe knowledge needs questionnaire of respiratory rehabilitation training for in-service healthcare workers included progress in respiratory rehabilitation, physiological basis and diagnosis of chronic lung diseases, individualized assessment and management principles of chronic lung diseases, treatment of chronic lung diseases and guideline-recommended medications, oxygen therapy, exercise assessment and exercise prescription, tobacco addiction and cessation, nutritional assessment and weight management, psychological screening and intervention, self-management and health behavior promotion, and effectiveness assessment of respiratory rehabilitation, totaling eleven dimensions and 52 items. The total Cronbach's α was 0.921, the total folded half reliability was 0.904, the total retest reliability was 0.922, and the Cronbach's α of each dimension ranged from 0.909 to 0.953; the folded half reliabilities ranged from 0.882 to 0.924, and the retest reliabilities ranged from 0.908 to 0.950. The validity of the questionnaire's content was 0.902. In exploratory factor analysis, eleven metric factors were extracted, and their cumulative variance contribution rate was 75.324%. ConclusionThe questionnaire on the knowledge needs of respiratory rehabilitation training for in-service healthcare workers is reliable and valid, and can be used to assess the knowledge needs of healthcare workers participating in in-service training in respiratory rehabilitation.