This study investigated gasoline–biodiesel blended fuel (GB) subjected to a fuel spray development process on macroscopic and microscopic scales. The four tested fuels were neat gasoline and gasoline containing biodiesel (5%, 20%, and 40% by volume) at three different ratios. The initial spray near the nozzle revealed that the spray penetration and spray tip velocity both decreased with decreasing biodiesel blending ratio. In addition, the different spray tip velocities at the start of spraying result in different atomization regimes between the fuels. The GB fuels with a low biodiesel blending ratio were disadvantaged in terms of spray atomization due to their lower spray penetration and tip velocity. The macroscopic spray penetration changes were similar to those observed in the microscopic spray. The fuel with the lower biodiesel blending ratio had a larger spray cone angle, indicating increased radial spray dispersion.