Background: Web-based questionnaire (WBQ) surveys are popular, but the quality of reporting WBQ survey research is uneven and unsatisfactory worldwide. Education and training on WBQ methodology may be necessary. However, the current knowledge, attitudes, and practices (KAP) of its methodology remain unknown. Objective: We investigated the KAP of WBQ methodology among Chinese health workers for the first time to clarify the possible reasons for the unsatisfactory reporting quality of WBQ survey research from China’s experience, aiming to provide a basis for improvement. Methods: We developed a structured WBQ based on the current recommendations and knowledge and investigated 458 health workers from June 7 to July 6, 2022. A total of 381 valid questionnaires were analyzed after data processing. We defined 50% and 75% as “qualified” and “satisfactory” in knowledge and practice topics to describe the results and analyzed the basic characteristics of the participants who had difficulties in conducting WBQ survey research. Results: A total of 215 (56.4%) participants had used WBQs for investigation, mostly more than 2 times (88.3%), but only 95 (44.2%) of them had ever received methodological training. A total of 134 (62.3%) users believed that WBQs were practical, but 126 (58.6%) had doubts about the reliability of the results. Most of the knowledge and practice topics did not reach a satisfactory level, and some even did not reach a qualified level. A total of 95 (44.2%)-136 (63.3%) of the users had reported difficulties in conducting WBQ survey research, and different participants could have different difficulties according to their characteristics. In addition, 191 (88.8%) users believed training was necessary. Conclusions: We found that Chinese health workers seriously underestimated and neglected the importance of the WBQ methodology, which may be an important reason for the reduced reporting quality of WBQ survey research. Medical educators need to strengthen methodological training on WBQs, which may help to improve the quality of WBQ survey research.