The current clinical protocol to conduct a bacterial antibiotic susceptibility test (AST) requires at least 18 hours, and cannot be accomplished during a single visit for patients. Here, a new method based on the technique of CRISPR-Cas12a is utilized to accomplish a bacterial genotypic AST within one hour with good accuracy. Two amplification approaches are employed and compared: (1) enriching the bacterial concentration by culturing in growth media; and (2) amplifying target DNA from raw samples by recombinase polymerase amplification (RPA). The results show that CRISPR combined with RPA can rapidly and accurately provide a bacterial genotypic AST of urine samples with urinary tract infections for precise antibiotic treatment. As such, this technology could open a new class of rapid bacterial genotypic AST for various infectious diseases.