Various research tools have been used for in vitro detection of sperm chemotaxis. However, they are typically poor in maintenance of gradient stability, not to mention their low efficiency. Microfluidic device offers a new experimental platform for better control over chemical concentration gradient than traditional ones. In the present study, an easy-handle diffusion-based microfluidic chip was established. This device allowed for conduction of three parallel experiments on the same chip, and improved the performance of sperm chemotaxis research. In such a chip, there were six channels surrounding a hexagonal pool. The channels are connected to the hexagon by microchannels. Firstly, the fluid flow in the system was characterized; secondly, fluorescein solution was used to calibrate gradient profiles formed in the central hexagon; thirdly, sperm behavior was observed under two concentration gradients of progesterone (100 pM and 1 mM, respectively) as a validation of the device. Significant differences in chemotactic parameters were recognized between experimental and control groups (p < 0.05). Compared with control group, sperm motility was greatly enhanced in 1 mM group (p < 0.05), but no significant difference was found in 100 pM group. In conclusion, we proposed a microfluidic device for the study of sperm chemotaxis that was capable of generating multi-channel gradients on a chip and would help reduce experimental errors and save time in experiment.