Mixing of binary or multi-component fluid streams is difficult in microchannels because they rely on diffusion during a limited mixing length. Therefore, obstacles applied and placed in the microchannel need to be used to try to disrupt the flow and reduce the diffusion path. In this study, finding a better layout for such obstacles embedded in Y-type microchannel is important to enhance flow mixing and will be executed at Reynolds numbers ranging from 0.5 to 60. These parameters of layout, such as number, horizontal spacing distance and angle of arrangement for cylinders embedded in the channel, as well as an inlet Reynolds number ratio, are worthy of study because they directly influence mixing and have rarely been previously addressed. The useful numerical results confirmed by our experiment will be addressed in this paper. Generally speaking, a larger number of cylinders, and a smaller horizontal spacing distance between them, normally correspond to a stronger flow mixing. In the case studied herein, five cylinders, arranged in a V with an angle of 90° at the base of the V, and the horizontal spacing distance between pairs of cylinders of 100 μm, is a better choice for providing enhanced flow mixing. In addition, the Reynolds number ratio of ten is suggested because it induces a more intensive lateral convection and produces enhanced flow mixing. Placing obstacles or textures in the microchannel is a significant method for mixing in microfluidic devices because of its simple prototype and ease of fabrication, and the results could provide useful information for future optimal design of these devices.