Objective To explore the solution strategies for the "difficult implementation" of drug-resistant orga-nism prevention and control measures in intensive care unit (ICU), and evaluate the effectiveness. Methods Patients admitted to the ICU of a tertiary first-class hospital from April 2022 to February 2024 were selected as the research subjects. The prospective, before and after comparison design was adopted, April 2022 to June 2023 and July 2023 to February 2024 were as the control stage and intervention stage, respectively. The control group implemented bundled prevention and control measures and routine management, the intervention group established a three-tier healthcare-associated infection (HAI) management structure (department internal management-section/group-employee) on the basis of implementing measures of control group, the implementation of various infection control measures was promoted. The 1∶1 propensity score matching (PSM) was used to evaluate the incidence of HAI and colonization rate of carbapenem-resistant organism (CRO) before and after intervention. Results The intervention group used 6.10 and 7.26 times more alcohol-based hand rub and hand sanitizers, 7.18 and 5.69 times more chlorine-containing disinfectant effervescent tablets and disinfectant wipes, as well as 2.76 times more isolation gowns respectively than the control group, the isolation order issuance rate increased from 62.89% to 89.27%, differences were all statistically significant (all P<0.001). Before PSM, the risk of acquiring CRO, carbapenem-resistant Acinetobacter baumannii (CRAB), and carbapenem-resistant Klebsiella pneumoniae (CRKP) in intervention group was 0.61, 0.67, and 0.80 times that of before intervention, respectively, except for CRKP (P=0.245), others were all with statistically significant differences (all P<0.05); After PSM, the risk of acquiring CRO, CRAB, and CRKP in the intervention group was 0.97, 0.92, and 0.97 times that of before intervention, respectively, all were with statistically significant differences (all P<0.05). Conclusion After implementing the three-tier management on infection prevention and control in department, the implementation of ICU isolation measures has significantly improved, and the incidences of CRO HAI and colonization have decreased.