The fossil fuel is consumed faster than last century. However, the fossil fuel reserves might be depleted due to its limited resources. Many researchers have started to seek alternative of fossil fuels for vehicles. Biofuels are regarded as a shorten-solution which synthesizes some additive fuel from bio-materials into fossil fuel to reduce fossil fuel consumption and can be applied on original fuel supplying system of vehicles that becomes another advantage. This paper focuses on studying the operation characteristics of using gasohol biofuel in a production motorcycle engine.Engine experiments with various test conditions are designed to investigate the operation characteristics with different concentration of ethanol. The ethanol concentrations considered in this paper include 10%, 20%, 50%, 65%, and 80% by weight, which are then compared with the results from pure gasoline. The engine test conditions cover engine speed ranging from 2000 to 7000rpm and with different operation loads. Experimental results are analyzed on maximum engine torque, cylinder pressure, combustion rate, fuel consumption, exhaust temperature, and emissions. Consequently, experimental results show that the gasohol biofuel performs similar operation characteristics as compared with pure gasoline, which means that the gasohol biofuels can be employed as an alternative fuel for vehicles.