Biodiesel derived from vegetable oil is now widely considered as an alternative fuel for CI engine especially in agricultural applications since the properties are closer to diesel than its raw material. However, the density and viscosity of biodiesel are higher than those of diesel fuel. This may affect the fuel spray characteristics, leading to the inefficiently of performance and exhaust emissions. In this paper, the effects of injection pressure and ambient gas temperature on spray characteristics were investigated by mean of the spray tip penetration. The experiment was taken on special made combustion chamber equipped with a single hole injector on common-rail system. Series of Shadowgraph images obtained from palm biodiesel and diesel were evaluated and compared. The results show that the spray tip penetration increases when the injection pressure is increased due to the increase of initial injection momentum. The results also show that the spray tip penetration of biodiesel is shorter and the injection delay is longer than those of diesel due to its high viscosity and density. The ambient temperature also affects the spray tip penetration. The increase of ambient temperature induces the shortening of spray tip penetration as the density of fuel is decreased.