Objective To perform epidemiological investigate on the suspected outbreak of blood stream infection (BSI) in orthopaedic patients after operation in a hospital, so as to provide reference for the prevention and control strategy of healthcare-associated infection (HAI). Methods Epidemiological data of patients with chill and high fever after operation in this hospital from July 16 to 18, 2020 were collected, bacteria isolated from patients, as well as their surroundings and articles were performed antimicrobial susceptibility testing, multilocus sequence typing (MLST) was used for homology analysis. Results A total of 4 cases of healthcare-associated blood stream infection caused by Enterobacter cloacae (E. cloacae) were found in department of orthopedics, the onset time was concentrated and antimicrobial susceptibility testing results of pathogens were similar. A total of 61 environmental specimens were collected, 26 of which were isolated bacteria, with a positive rate of 42.62%, 8 isoosmotic rinsing solution with batch number of 2020071* * were isolated E. cloacae, 2 isoosmotic rinsing solution with batch number of 2020061* * were not isolated E. cloacae. MLST results of 12 strains of E. cloacae were ST1190 (1 strain), ST531 (2 strains), ST547 (2 strains), ST290 (1 strain), ST777 (3 strains), ST422 (1 strain), ST1032 (1 strain) and ST717 (1 strain), there are homologous E. cloacae. Conclusion The suspected outbreak of post-operative BSI in orthopaedic patients in this hospital may be related to the contamination of a new batch of isoosmotic rinsing solution used for operation during transportation or production. Hospitals should strengthen the supervision of disposable foreign medical supplies, regularly sample sterile items, and use them only after passing the test, so as to reduce the risk of exogenous infection and prevent the outbreak of BSI events.