Uropathogenic Escherichia coli(UPECs)is a leading cause for urinary tract infections (UTI), accounting for 70-90% of community or hospital-acquired bacterial infections owing to high recurrence,imprecision in diagnosis and management, and increasing prevalence of antibiotic resistance. Current methods for clinical UPECs detection still rely on labor-intensive urine cultures that impede rapid and accurate diagnosis for timely UTI therapeutic management. Herein, we developed a first-in-class near-infrared (NIR) UPECsfluorescent probe (NO-AH) capable of specifically targetingUPECsthrough itscollaborativeresponse to bacterial enzymes,enabling locoregional imaging of UTIs both in vitroand in vivo.Our NO-AH probe incorporatesa dual protease activatable moiety, which first reactswithOmpT, an endopeptidase abundantly present on outer membrane of UPECs, releasing an intermediate aminoacid residue conjugatedwith a NIR hemicyanine fluorophore. Suchliberated fragment would be subsequentlyrecognized by aminopeptidase (APN) within periplasm of UPECs, activating localized fluorescence forprecise imagingof UTIs in complex living environments.The peculiar specificity and selectivity of NO-AH, facilitated by the collaborative action of bacterial enzymes, features a timely and accurate identification of UPECs-infected UTIs, which could overcomemisdiagnosis inconventional urine tests,thus opening new avenues towards reliable UTI diagnosis and personalized antimicrobial therapy management.