Objective To explore the current situation of healthcare-associated infection (HAI) in patients in emergency rescue room and observation room, and analyze the causes. Methods Targeted surveillance was performed on patients in emergency rescue room and observation room of a tertiary first-class hospital in Xuzhou City from January 1 to December 31, 2023 using prospective survey method. HAI, antimicrobial use, as well as invasive procedures and related infection rates were analyzed. Results The incidence of HAI in 540 patients was 0.19% (n=1), and the daily incidence of HAI was 0.54‰ (1/1 841). The utilization rates of invasive ventilators, central venous catheters, and indwelling urinary catheters were 6.79% (125/1 841), 1.09% (20/1 841), and 2.93% (54/1 841), respectively. The incidences of ventilator-associated pneumonia, central line-associated bloodstream infection, and catheter-associated urinary tract infection were 8.00‰ (1/125), 0 (0/20), and 0 (0/54), respectively. Pathogen detection rate before antimicrobial treatment was 99.22% (382/385). Conclusion The incidence of HAI in patients in emergency rescue room and observation room is low, the retention time of patients in rescue room is short, and pathogen detection of specimens before antimicrobial treatment is high. Patients in emergency rescue room and observation room are at high risk of HAI. It is necessary to carry out targeted surveillance on HAI to reduce its incidence.