This study presents a microfluidic approach for the rapid analysis of bacterial chemotaxis in response to chemical gradients. The diffusional mixing of laminar flow continuously generates a stable chemical gradient in a microfluidic device. For the proof of concept, we have investigated the effects of the attractant peptone and repellent trichloroethylene (TCE) on chemotactic responses of wild type Pseudomonas aeruginosa PAO1 and chemotactic mutant PC4. The microfluidic method clearly demonstrates that P. aeruginosa PAO1 is attracted to peptone and repelled from TCE, whereas PC4 shows non-chemotactic behavior. In addition, the analysis of PAO1 chemotaxis on 20 amino acids revealed the effective concentration range of each amino acid as a chemoeffector. Not only does the microfluidic approach facilitate the quantitative information of chemotaxis, which gives an insight into understanding the mechanism of P. aeruginosa motility, but it also provides a useful tool for the rapid monitoring of bacterial chemotaxis in a reproducible experimental manner.