Objective To observe the effectiveness of fluorescence labeling-based assay bundle intervention in the prevention and control of multidrug-resistant organism (MDRO) infection. Methods Patients who were detected MDRO in a hospital from January to December 2022 were selected as the research subjects. MDRO monitoring data and implementation status of prevention and control measures were collected. Fluorescence labeling assay was adopted to monitor the cleaning and disinfection effectiveness of the surrounding object surface of the bed units. Based on the bundled prevention and control measures as well as management mode of the pre-intervention group, the post-intervention group implemented enhanced rectification measures for the problems found by the pre-intervention group. Changes in relevant indicators between January-June 2022 (before intervention) and July-December 2022 (after intervention) were compared. Results There were 136 MDRO-infected patients in the pre-intervention group, 208 MDRO strains were detected and 10 healthcare-associated infection (HAI) occurred. There were 128 MDRO-infected patients in the post-intervention group, 198 MDRO strains were detected and 9 HAI occurred. After intervention, the total detection rates of methicillin-resistant Staphylococcus aureus (MRSA), carbapenem-resistant Acinetobacter baumannii (CRAB), and total MDRO from patients decreased significantly compared to before intervention (all P < 0.05). After intervention, the detection rates of MRSA, carbapenem-resistant Pseudomonas aeruginosa (CRPA), CRAB, and total MDRO from the surrounding object surface were all lower than those before intervention (all P < 0.05). The detection rate of MDRO from surrounding object surface before intervention was 34.52%, which showed a decreased trend after intervention (P < 0.05). The clearance rate of fluorescent labeled markers before intervention was 41.84%, which showed an upward trend after implementing intervention measures (from July to December), and increased to 85.00% at the end of intervention (November-December). The comp-liance rates of issuing isolation medical orders, placing isolation labels, using medical supplies exclusively, and correctly handling medical waste after intervention have all increased compared to before intervention (all P < 0.05). Conclusion Adopting fluorescence labeling-based assay bundle intervention can effectively improve the effectiveness of MDRO infection prevention and control.