Objective To explore the application effect of risk assessment combined with plan-do-check-action (PDCA)cycle in the prevention and control of multidrug-resistant organism (MDRO) infection. Methods Relevant data of targeted monitoring on MDROs of "five categories and seven types" in a tertiary first-class hospital from July 2019 to June 2020 were selected. The pre-intervention stage was from July to December, 2019, and the post-intervention stage was from January to June, 2020. Based on risk assessment results in 2019, the PDCA cycle method was introduced to intervene and manage the detection of MDROs and the implementation of prevention and control measures. The detection of MDROs and the implementation rate of prevention and control measures before and after the intervention were compared. Results A total of 124 and 129 patients before and after the intervention were included respectively. After intervention, the pathogen detection rate before antimicrobial therapy was higher than before intervention (39.43% vs 33.13%, P < 0.001). The total implementation rate of MDRO prevention and control measures after implementing risk assessment combined with PDCA cycle intervention was 82.83%, which was higher than before the intervention (61.75%), with statistically significant difference (P < 0.05). After intervention, the implementation rates of 8 measures increased with statistically significant differences (all P < 0.05) compared to before intervention, including awareness of MDRO prevention and control measures, critical value reports, giving isolation medical orders, fluorescent monitoring on cleaning and disinfection of bed units, hand hygiene before and after operation, early handover about MDRO, notification of receiving departments to take isolation measures, and final disinfection. The detection rate of MDROs after intervention was 8.08%, which was lower than before intervention (10.31%), with statistically significant difference (P < 0.05). Detection rate of carbapenem-resistant Acinetobacter baumannii (CRAB) decreased from 60.78% to 48.13%, and detection rate of multidrug-resistant (MDR)/extensively drug-resistant Pseudomonas aeruginosa (XDR-PA) decreased from 35.35% to 22.90% after intervention, with both differences statistically significant (both P < 0.05). Conclusion Risk assessment combined with the PDCA cycle can improve the implementation rate of MDRO prevention and control measures, so that the MDRO detection rate can be reduced even with increased pathogenicity detection rate before antimicrobial therapy.