The object of this work is to analyze the macroscopic spray performance and atomization characteristics between diesel and biodiesel fuels. In this study, the effects of mixture ratios of biodiesel fuel on the spray tip penetration, fuel injection rate, spray cone angle, and the atomization characteristics such as droplet size, droplets distribution, and spray arrival time according to the axial distance were investigated at various injection parameters. It is revealed that the injection rate is more affected by injection pressure than mixture ratio. And, the spray development process is closely matched between diesel and biodiesel fuels. However, the droplet atomization characteristics of biodiesel shows deteriorated results as the mixture ratio of biodiesel increased because of the high viscosity and density.