Objective To investigate the economic burden of healthcare-associated infection (HAI) caused by multidrug-resistant organism (MDRO) in general intensive care unit(GICU), analyze the cost-benefit of prevention and control of MDRO HAI. Methods HAI in patients in a GICU from 2016 to 2017 were investigated retrospectively, 2016 and 2017 was as pre- and post-implementation group of infection prevention and control measures respectively, incidence of MDRO infection in two groups of patients was compared. A matched case-control study was used to compare the hospitalization expense and length of hospital stay between patients in MDRO HAI group and non-infection group, economic loss caused by MDRO HAI was evaluated, cost-benefit of prevention and control was analyzed. Results Incidence of MDRO HAI decreased from 4.4% to 1.9% after the implementation of HAI prevention and control measures. Compared with non-infected patients, hospitalization expense of MDRO-infected patients increased by 116 147.0 Yuan and length of hospital stay extended by 26.0 days. In 2016 and 2017, direct economic loss caused by MDRO HAI were 8 246 437.0 and 3 716 704.0 Yuan respectively, the profit loss due to the decrease of admission of patients were 232 328.2 and 90 561.1 Yuan respectively, and charge for loss of working time due to extension of length of hospital stay were 122 389.8 and 59 238.4 Yuan respectively. In 2017, the investment in prevention and control measures increased by 110 469.0 Yuan, direct and indirect economic loss reduced by 4 734 651.5 Yuan, ratio of benefit to cost was 42.9:1; economic loss of hospital reduced by 141 767.1 Yuan, ratio of benefit to cost was 1.3:1. Conclusion Effective implementation of prevention and control of HAI can not only reduce the incidence of HAI, but also bring huge social and economic benefit.