Microfluidic chemotaxis analysis requires stable and reproducible formation of chemical gradient with low batch-to-batch variation. Dimensional uniformity of a junction channel plays a key role in diffusion-based gradient generation. Conventional methods for the analysis of bacterial chemotaxis are limited in stability and reproducibility of the chemical gradient. Here, we report a microfabrication method employing plasma enhanced chemical vapor deposition for silicon oxide deposition and consecutive reactive ion etching processes to reduce the experimental errors of chemotaxis analysis occurring from dimensional inaccuracy of the junction channel. This approach produces precise dimensional junction channels with below 1% variation. Thus, we confirm the reproducibility of chemotaxis analysis and obtain the chemotaxis index of Pseudomonas aeruginosa PAO1 with bacterial chemoeffectors.