The purpose of this study is to evaluate the effect of online teaching of medical microbiology course in Dalian Medical University, find and summarize the problems in online teaching in time, and provide a basis for teaching curriculum reform. The examination paper of 641 students who participated in the online teaching and examination of medical microbiology course in 2021 was analyzed by using the network question bank and examination evaluation system of Dalian Medical University, including the accuracy rate of each chapter of the paper, difficulty coefficient, reliability and discrimination. The results showed that the score of this exam was normal distribution, the accuracy of each chapter was between 70% and 90%, the average score of the exam was 72.28, the pass rate was 85%, and the excellent rate was 4%. The overall quality of the examination paper was good.The difficulty of the examination paper was slightly easier, and the difficulty coefficient was 0.72. The discrimination was 0.29, the reliability was 0.9. There was no significant difference in the final exam scores between online teaching students in 2021 and offline teaching students in 2019 (P>0.05). This study confirms that the overall quality of the online examination paper is good, which can objectively reflect the teaching quality and students’ academic level. The online teaching effect is basically satisfied. This study also compares the difficulty coefficient and differentiation of each question type in the test paper to provide the basis and reference for the development of scientific and reasonable online examination papers in the future.