This research aims to evaluate the life cycle of the impact of using bioethanol in gasoline engines and the emission of greenhouse gases in each of the above stages. The production of bioethanol was obtained from the used biomass in a retention time of five days. In this research, the engine test was done with a two-stroke, single-cylinder engine. Performance parameters (braking power, brake-specific fuel consumption, and engine braking efficiency) and emission parameters (carbon dioxide emission, carbon monoxide emission, and nitrogen oxide emission) were recorded. Then, the list of power production cycles (to produce 1 kW of power) was prepared. Next, by applying the ISO14040 standard and the IMPACT2002+ assessment method, the life cycle assessment was performed with SIMAPRO software. According to the results obtained from the engine's performance test stage, increasing the bioethanol percentage in gasoline fuel reduced the amount of power production. Also, increasing the rate of bioethanol in gasoline fuel increased the consumption of special brake fuel. Further, with the increase in the percentage of bioethanol in gasoline fuel, from zero to five percent, the braking thermal efficiency of the engine decreased and increased from five to seven percent. According to the results obtained from the engine emission study, increasing the percentage of bioethanol from zero to two percent increased the emission of carbon monoxide, and from 2 to seven percent decreased the emission of carbon monoxide.