Rapid screening platforms for antibiotic susceptibility testing (AST) are important in inhibiting bacterial resistance in clinical practice. Herein, a rapid screening platform is reported for AST, which is based on nanofiber membrane enrichment bacteria-assisted cell counting Kit-8 (CCK8) colorimetry. The absorbance of CCK8 formazan has a linear relationship with the number of bacteria. The interference of antibiotics in the absorbance of CCK8 formazan could be eliminated by separating planktonic bacteria from the culture medium using nanofiber membranes. The total detection time is 7-9 h, using the new screening platform, which is significantly shorter than that with the traditional method, and the limit of detection of this method is 10 CFU mL-1. The evaluation results of antibiotic susceptibility are identical when using the new screening method and traditional methods. This method meets the definition of "rapid testing" for antibiotic susceptibility by most microbiologists. Furthermore, the new screening platform for antibiotic susceptibility testing ability in vitro was proved using E. coli in urine and blood, and S. aureus in wound fluid as practical samples. All the results showed that the new screening platform is a promising method for rapid antibiotic susceptibility testing in vitro.