This paper investigated the effects of gas-to-liquid (GTL), biodiesel, and fuel injection parameters such as injection pressure and ambient pressure on the spray macroscopic characteristics, spray behavior, fuel injection rate, and combustion characteristics in a diesel engine. In this experiment, the spray characteristics of GTL as an alternative fuel, including spray tip penetration, spray cone angle, injection rate, and spray development images, were compared with those of biodiesel fuel derived from soybean oil and conventional diesel fuel. Based on the experimental results the fuel spray tip penetration of GTL fuel showed short penetration compared to those of biodiesel and conventional diesel fuel, and the spray cone angle was larger than those of biodiesel and diesel fuel. GTL fuel had shorter injection duration than that of biodiesel and diesel fuel, and the peak injection rate of GTL increased with injection pressure. The spray development images showed that the spray images of GTL fuel were slightly shorter than those of biodiesel, and spray characteristics were more affected by the ambient and injection pressures than by fuel properties. In addition, the combustion characteristics showed that GTL fuel has short ignition delay characteristics, and it prevents a rapid increase in the cylinder pressure. Therefore, the maximum pressure of GTL is lower than that of diesel and biodiesel fuel.