Objective To construct a knowledge graph of healthcare-associated infection (HAI) prevention and control course, and evaluate its usability. Methods Based on the constructivist learning theory and the analyze-design-develop-implement-evaluate (ADDIE) model, knowledge from various sources such as books, guidelines, and literature related to HAI prevention and control were integrated. The knowledge graph of HAI prevention and control course were designed and constructed with the support of knowledge graph technology in Chaoxing Fanya platform. Thirty medical students were selected by convenience sampling method to try out the course knowledge graph. System usability scale and usage effect questionnaire were filled out to evaluate the usability of the knowledge graph. Results The knowledge graph of HAI prevention and control course contained 379 knowledge points asso-ciated with 520 test questions and 56 learning resources. After testing, the total score ([70.50±12.20] points) was obtained for the usability of the knowledge graph. Among the four dimensions of the usage effect agreement questionnaire, satisfaction, learning attitude, learning ability, and learning resource support accounted for 93.33%-96.67%, 90.00%, 93.33%-96.67%, and 83.33%-90.00%, respectively, with a high overall satisfaction rate. Conclusion The knowledge graph of HAI prevention and control course has good usability, which can realize students’ personalized independent learning and improve their learning efficiency.