OBJECTIVE To investigate the factors responsible for Pseudomonas aeruginosa nosocomial cross-infection in the intensive care unit (ICU) and provide effective measure for the prevention and management. METHODS The homology of 7 Pseudomonas aeruginosa strains isolated from the patients in the ICU and the environment was examined by biological, serological, drug-resistance and plasmid analysis. RESULTS The results of serological and plasmid analyses had good consistency, which demonstrated that the 7 Pseudomonas aeruginosa strains originated from solution in the oxygen humidifier, with uniform serological and plasmid type, and belonged to the same clone with cross-infection by contact between the patients or nursing staff and the objects in the ICU. CONCLUSIONS Serological and plasmid analyses are more practical for identifying the sources of Pseudomonas aeruginosa cross-infection, which can be prevented by strict disinfection of the instrument and supervision of the nursing staff in the ICU.