The urinary tract infections by antibiotic-resistant bacteria have been a serious public health problem and increase the healthcare costs. The conventional technologies of diagnosis and antimicrobial susceptibility testing (AST) relying on multiple culture-based assays are time-consuming and labor-intensive and thus compel the empirical antimicrobial therapies to be prescribed, fueling the prevalence of antimicrobial resistance. Herein, we propose an all-in-one Escherichia coli viability assay in an enclosed 3D microwell array chip, termed digital β-d-glucuronidase (GUS)-AST assay. It employs GUS, a specific metabolism-related enzyme, to convert the presence of E. coli into bright fluorescence. The random distribution of single bacteria in microwell array enables to quantify the E. coli concentrations by counting the positive microwells. We incorporate the most probable number with digital quantification to lower the limit of detection and expand the dynamic range to 7 orders. The digital GUS-AST assay is able to indicate the potency of antibiotics and determine the minimum inhibitory concentrations. A streamlined procedure of urine removal, bacterial separation, and digital GUS-AST is established to perform the direct analysis of bacteria population in urine. The sample-to-result workflow can be finished in 4.5 h with a limit of detection of 39 CFU/mL. With further development for additional pathogens and multiple antibiotic conditions, the digital GUS-AST assay could help physicians to prescribe timely targeted therapies for better patient outcomes and the minimum emergence of resistant bacteria.