Objective To analyze the correct answering rate and factors affecting healthcare-associated infection (HAI) test questions among medical interns, and provide reference for formulating teaching plans. Methods All medical interns who participated in pre-job training and testing on HAI in a hospital from October 2020 to February 2024 were selected. Pre-internship training and in-class testing on HAI were conducted. After the internship ended, a class of students was randomly selected each year to directly take the written test using the same set of questions. The impact of test content, question types, majors, educational background, and the epidemic on the correct answering rate of pre-internship test was analyzed, and correct answering rate for tests before and after internships was compared. Results A total of 1 163 interns were assessed, out of which 48.75% obtained a correct answering rate of 90%-100% for test questions before the internship. Among the 10 test contents, the correct answering rates for questions about multidrug-resistant organism infection prevention and control were the lowest (［67.13±34.35］ %), among 3 types of questions, the correct answering rate for indefinite-choice questions was the lowest (［79.80±19.31］ %). The correct answering rates of interns majoring in clinical medicine (［90.49±12.32］ %) and nursing (［87.54±10.73］ %) were higher than those of other majors (［82.80±12.24］ %). The correct answering rate of undergraduate students (［89.05±11.29］ %) was higher than that of junior college student (［83.77±12.26］ %). The correct answering rate of interns during the epidemic period (［87.51±11.48］ %) was higher than that after the epidemic (［79.85±13.98］ %), and the correct answering rate after the internship (［81.89±14.78］ %) decreased compared with that before the internship (［92.99±10.48］ %). Differences were all statistically significant (all P<0.005). Conclusion Teachers can carry out targeted teaching plan reforms based on the factors affecting the correct answering rate (such as different majors, teaching content, and internship periods) to improve teaching quality.