Objective To evaluate the economic burden of healthcare-associated infection (HAI) in hospitalized patients in the general intensive care unit (ICU) by using the disease diagnosis-related group (DRG) method, and provide basis for optimizing HAI management strategies. Methods Clinical data of hospitalized patients in the general ICU of a tertiary first-class hospital from January 1, 2022 to December 31, 2024 were collected retrospectively, patients were divided into HAI group and non-HAI group based on whether they had HAI. The case mix index (CMI), hospitalization expense, and length of hospital stay between two groups of patients were compared and analyzed with DRG system. Results A total of 2 874 hospitalized patients in general ICU were included in analysis, out of which 201 had HAI, with HAI incidence of 6.99%. The CMI value of HAI group was higher than that of non-HAI group (6.48 vs 1.78), difference was statistically significant (P<0.001). The median hospitalization expense of HAI group (108 999.91 Yuan) increased by 65 329.05 Yuan compared with non-HAI group (43 670.86 Yuan), and the median length of hospital stay was extended by 19.00 days (29.00 vs 10.00 days), both with statistically significant differences (both P<0.001). Among the top ten DRGs with the highest incidence of HAI, FP1 (heart failure, shock with manipulation treatment) had the longest length of hospital stay and the largest increase in expense, which were 45.50 days and 511 539.67 Yuan, respectively. Bacteremia, catheter-associated urinary tract infection (CAUTI), central line-associated bloodstream infection (CLABSI), and ventilator-associated pneumonia (VAP) had a long length of hospital stay and expense consumption index, with length of hospitalization extension≥15 days and hospitalization expense increase>42 996 Yuan. MDRO infection brought about significant economic burden, patients in carbapenem-resistant Enterobacterales (CRE) HAI group had an extension of 25.00 day of hospital stay and an increase of 59 873.84 Yuan of expense. Hospitalized expense of patients with carbapenem-resistant Pseudomonas aeruginosa (CRPA) in HAI group was 2.25 times of non-HAI group. Conclusion There are significant differences in the economic burden caused by different DRGs, different infection types, and different MDRO infection. Based on DRG evaluation, the economic losses caused by HAI can be accurately quantified, which provide evidence-based support for prioritizing key prevention and control measures and optimizing resource investment.