This study was performed to investigate the effect of biodiesel derived from waste cooking oil on the spray behavior and macroscopic spray characteristics. To analyze quantitative characteristics of test fuels, injection quantity was measured at various injection pressures and the spray images of injected fuels in the pressurized chamber were obtained by using a high speed camera and image analysis system. Based on the measured spray images, the spray tip penetration and spray cone angle were investigated at various energizing timings and injection pressures. In this work, the experimental results showed that the injection quantity of waste cooking biodiesel indicated the higher quantities than diesel at high injection pressure. As the injection pressure was increased, the spray tip penetrations of biodiesel were higher value than diesel. The difference of penetration between biodiesel and conventional diesel fuel was reduced in accordance with the increase of injection pressure. Also, the spray angles of diesel were larger than that of biodiesel because diesel fuel has lower viscosity than biodiesel. In addition, the spray evolution processes of biodiesel fuel at various injection pressures and the elapsed time after the injection were compared to the conventional diesel fuel.