A compact disk-based enzyme linked immunosorbent assay (CD-ELISA) is used in a wide range of applications including cancer and human immunodeficiency virus testing, drug screening, and micro-organism identification. Bifurcation design is the most important structure for microfluidic channels in CD-ELISA. In this study, a bifurcation design feasible for mass production and suitable for applications over a wide range of rotational speeds is proposed. Simulations based on two-phase flow theories along with incompressible flow theories were used in this study to confirm the feasibility of the novel design. The factors that influenced the bifurcation ratio for microfluidic channels in CD-ELISA were also investigated. The geometric length for bifurcation, opening angles, and the bifurcation shape in the middle section were varied to investigate the effects of each factor on the bifurcation ratio. From the experimental results, the factors with the greatest influence on the bifurcation ratio were the geometry of the end face of the partitioning plate and the distance from the opening end. These factors can be used as controlling factors for the design of microfluidic channels.