The antibiotic susceptibility test (AST) is a general laboratory procedure for bacterial identification and characterization and can be utilized to determine effective antimicrobials for individual patients. Due to the low bacterial concentration, conventional AST usually requires a prolonged bacterial culture time and a labor-intensive sample pretreatment process. Therefore, it cannot perform timely diagnosis or treatment, which results in a high mortality rate for seriously infected patients. To address this problem, we developed a microfluidic microwell device integrating surface-enhanced Raman scattering (SERS) technology, or the so called the Microwell-SERS system, to enable a rapid and high-throughput AST. Our results show that the Microwell-SERS system can successfully encapsulate bacteria in a miniaturized microwell with a greatly increased effective bacteria concentration, resulting in a shorter bacterial culture time. By attaching a microchannel onto the microwell, a smooth liquid and air exchange can purify the surrounding buffer and isolate bacteria in an individual microwell for independent SERS measurement. For proof-of-concept, we demonstrated a 2 h AST on susceptible and resistant E. coli and S. aureus with a concentration of 103 CFU mL-1 in the Microwell-SERS system, whereas the previous SERS-AST method required 108 CFU mL-1 bacterial suspension droplets dispensing on a SERS substrate. Based on the above features, we envision that the Microwell-SERS system could achieve highly sensitive, label-free, bacteria detection and rapid AST to enable timely and accurate bacterial infection disease diagnosis.