Environmental and energy independence concerns have stimulated the development of an apparatus for alternative fuel. It estimates the ethanol content in the fuel in order to perform a reliable combustion. One means for measuring the ratio of ethanol present in the fuel tank is to provide a fuel composition sensor. However, such a fuel composition sensor increases the number of parts and causes the cost issues in motorcycles. We used an oxygen sensor disposed to the exhaust pipe to estimate the ethanol content without increasing the parts and costs.The common method of the estimation is the oxygen feed-back in stoichiometric air fuel ratio condition. Unfortunately, two-wheel vehicles are often operated in rich conditions and have less chance of stoichiometric condition. In this study, we used a one-liter four-cylinder motorcycle, and have developed a practical method to estimate the ethanol content even in the not-stoichiometric condition. A newly developed method uses two lambda variables (ratio of actual air-fuel-ratio to stoichiometric air-fuel-ratio) and provides more chance of the oxygen feedback for motorcycles. Using an engine dynamometer and a chassis dynamometer, we conducted some verification examinations for this method and the apparatus. The results showed the method's applicability from 0% to 100% ethanol content.