Bioluminescence technology has been widely used in the field of medical detection. The bioluminescentluxreporter system provides a non-invasive platform to monitor bacterial growth and expression in real time. This study aimed to establish a method for detecting bacterial adhesion on the surface of materials, including medical devices, by using recombinant bioluminescentPseudomonas aeruginosacontaining aluxreporter. By monitoring the growth and bioluminescent properties of the recombinant PAO1-luxstrain, the optimal test conditions for bacterial adhesion detectionin vitrowere determined to be as follows: an initial inoculation density of 105to 106CFU/mL, M9 medium at a pH 6.2, an adhesion time of 6 h, and the collection of adherent bacteria by ultrasonic cleaning. The traditional CFU counting method and the bioluminescence method were compared, and the applicability of the new method was verified by testing the adhesion of bacteria on the surface of various materials. The validated bioluminescent strains could serve as strong candidates to be used as bacterial detection tools in applications such as bacterial adhesion evaluation as well as supplements and alternatives to traditional microbiological testing procedures. In addition, this method has the potential to enable the study of bacterial adhesion on the surface of inanimate objects and living tissues. With the development of this method and its wide applicability, it is expected to become a standard method for the detection of bacterial adhesion and the screening of anti-adhesion materials.