ObjectiveTo explore the application of adenosine triphosphate (ATP) bioluminescence assay in evaluating cleaning quality of medical devices.  MethodsManual and machine cleaned medical devices were randomly selected from central sterile supply department of a hospital in 2011-2013, cleaning quality was detected with ATP bioluminescence assay, relative light unit (RLU) value was determined to assess the cleaning quality. ResultsA total of 460 pieces of cleaned devices were detected in 2011-2013, 404 (87.83%) were qualified. The qualified rates of manual  cleaning and machine cleaning were 70.73% and 94.07% respectively, the qualified rates of general surgical devices and lumen devices were 90.71% and 81.76% respectively, there were significant differences in qualified rates of different cleaning methods and different categories of medical devices (both P<0.01). The qualified rates of manual  cleaning in 2011-2013 were 32.35%, 79.63%, and 94.29% respectively, machine cleaning were 79.45%, 98.15%, and 98.08% respectively；differences in qualified rates of manual  cleaning and machine cleaning in different years were significant (all P<0.01). ConclusionATP bioluminescence assay can be used for evaluating cleaning quality of medical devices.