Objective To perform active screening on carbapenem-resistant Gram-negative bacilli (CR-GNB), carry out centralized resettlement for patients according to the screening results, evaluate the efficacy of intervention measures, and provide scientific basis for the prevention and control of healthcare-associated infection(HAI) due to CR-GNB. Methods From January 2017 to December 2018, patients who were admitted to key departments of a pediatric hospital (neonatal room, neonatal intensive care unit [NICU], pediatric intensive care unit [PICU], department of hematology) were selected as the research objects, all patients were performed HAI real-time monitoring. In 2017, active screening on CR-GNB (throat swab and anal swab) was performed for hospitalized patients 48 hours after admission and once a week during hospitalization. From January 2018, centralized resettlement measures were carried out for patients with positive CR-GNB in active screening and clinically delivered specimen detection, changes of active screening rate and colonization rate of CR-GNB as well as centralized resettlement rate and HAI rate were compared. Results Active screening rates of throat swab and anal swab in 2018 was higher than those in 2017 (P < 0.05). The highest centralized resettlement rate was in neonatal room (97.8%), followed by NICU (88.9%), PICU was the lowest (47.7%). In 2018, colonization rate of CR-GNB in key departments showed a downward trend after 3 and 7 days of hospitalization (P < 0.05). HAI rate of CR-GNB in 2018 decreased compared with that in 2017 (P < 0.05), carbapenem-resistant Enterobacterales HAI rate in neonatal room and NICU decreased significantly, HAI rates of carbapenem-resistant Acinetobacter baumannii (CRAB) and carbapenem-resistant Pseudomonas aegurinosa (CRPA) in patients in PICU decreased significantly. Carbapenem-resistant Klebsiella pneumoniae (CRKP) was the main pathogen in active screening of colonized bacteria and pathogens of CR-GNB HAI (44.0% vs 51.7%). Conclusion Active screening of CR-GNB combined with centralized resettlement intervention for patients can reduce the colonization rate of CR-GNB and effectively reduce HAI rate.