The separation of bacteria from infected blood is a crucial step for downstream bacterial detection and analysis. However, the extremely low bacterial concentration and high proportion of blood cells present significant challenges for effective separation. In this study, we present a microfluidic-based approach utilizing a sequential trench well structure that leverages the sedimentation-based separation process to effectively isolate bacteria from whole blood. Experimental results demonstrated that the system achieved an RBC removal rate of over 99.99%, while maintaining a bacterial recovery rate of up to nearly 78%. We utilized the separation platform to isolate bacteria from whole blood samples and applied separated bacterial samples to the image-based antimicrobial susceptibility testing (AST) process, reducing the conventional AST procedure by up to 42.18 hours. Our separation technology provides a simple and effective method for isolating bacteria from blood samples, with potential applications in infection diagnostics and microbiological analysis.