Pseudomonas aeruginosa (P. aeruginosa) is one of the most intractable multidrug-resistant bacteria of nosocomial infections. The conventional detection methods for P. aeruginosa are time-consuming or low detection sensitivity. Here, a novel enzyme-free electrochemical biosensor was constructed to detect P. aeruginosa rapidly and sensitively. Firstly, the ZrMOF with large surface area was synthesized, which offers excellent adsorption. Further, it was connected with a specific amount of Cu to synthesize Cu-ZrMOF with high catalytic activity. Then the Cu-ZrMOF@Aptamer@DNA nanocomposite was composed and served as the signal probe to catalyse the decomposition of HO. Moreover, high conductive Super P was introduced to increase the electron transfer for satisfactory detection sensitivity. The proposed biosensor was constructed and used to quantify P. aeruginosa with a wide linearity range of 10-10 CFU mL and a low limit of detection of 2 CFU mL (S/N = 3). Compared with conventional methods, the new method of present biosensor is more sensitive, and less time-consuming (only within 120 min). The analytical performance evaluation indicated that the biosensor exhibits good reproducibility and specificity. Finally, the biosensor was successfully applied to quantify P. aeruginosa in spiked urine samples. These results show that the proposed electrochemical biosensor might be a potential laboratory tool for detecting P. aeruginosa in the clinic.