The ability to appraise antibacterial potencies of surface-immobilized bactericidal polymers is still a major challenge in the engineering of antibacterial surfaces to combat hospital-acquired (nosocomial) infections. In this work, we fabricated a microfluidic platform with gradiently immobilized bactericidal polymers to enable the rapid appraisal of antibacterial potencies by in situ live/dead staining of bacteria. To this end, a variety of synthetic quaternary polymers, named QPEI-C-1, QPEI-C-6, QPEI-C-8, and QPEI-C-10, were gradiently immobilized in microfluidic channels, and their surface densities at different distances along the channels were quantified by using fluorescein-labeled polymers. We found that the surface densities of quaternary polymers could be well-tuned, and the length of the channel, resulting in a 50% reduction of live bacteria (L-50), can be used to appraise the antibacterial potency of each bactericidal polymer. For instance, the L-50 values of QPEI-C-6(-), QPEI-C-8(-), and QPEI-C-10-modified channels against Escherichia coli were 35.5, 44.7, and 49.2 mm, respectively, indicating that QPEI-C-10 exerted the most potent antibacterial efficacy. More importantly, this microfluidic platform enabled the rapid discrimination of antibacterial potencies of polymers (e.g., QPEI-C-8, and QPEIC-C-10) while the conventional live/dead staining method found no significant difference. This work provides a powerful toolkit by combining advances of microfluidic systems and polymer science for the rapid screening of antibacterial coatings, which would find applications in surface modification of medical devices to combat bacterial infections.