Objective To explore the value of analysis on the incidence of healthcare-associated infection (HAI) based on disease diagnosis-related grouping (DRG), case mix index (CMI), and relative weight (RW). Methods All discharged cases, DRG and HAI status in a tertiary first-class general hospital from January 1 to December 31, 2023 were analyzed retrospectively. Incidences of HAI in different departments were adjusted and compared by CMI. Incidences of HAI in different DRG groups were adjusted by RW. Results Among the 47 695 cases included in the analysis, 757 were HAI cases, including 225 DRG groups. The department of critical care medicine had the highest incidence of HAI (11.98%). After CMI adjustment, departments with higher incidence of HAI were mainly the department of respiratory and critical care medicine (3.96%), department of critical care medicine (3.04%), and department of neurology (2.85%), et al. DRG groups with the top five high incidence of HAI were AH11 (tracheotomy and with ventilator support ≥96 hours or extracorporeal membrane oxygenation ［ECMO］, accompanied by major complications and comorbidity ［MCC］, 50.00%), BC29 (ventricular shunt and revision surgery, 31.43%), BB21 (craniotomy other than trauma, accompanied by MCC, 27.56%), BB11 (craniotomy of brain trauma, accompanied by MCC, 26.32%), and GB1A (major surgery of esophagus, stomach, and duodenum, accompanied by major or moderate complications and comorbidity, 16.00%). After RW adjustment, the DRG groups with the top five high incidence of HAI were ES21 (respiratory system infection/inflammation, accompanied by MCC, 5.89%), BR21 (cerebral ischemic disease, accompanied by MCC, 5.17%), FR11 (heart failure, shock, accompanied by MCC, 4.80%), BC29 (4.57%) and AH11 (3.57%). Conclusion Analyzing the incidence of HAI based on CMI and RW can help to identify key departments and disease groups for infection prevention and control, and provide reference for precise prevention and control of HAI in the new era.