Biogas is one of promising renewable fuel produced from several numbers of organic substrates. In fact, biomass as the organic matters has an opportunity to generate a cheaper and cleaner energy than using other resources. Therefore, many researchers have been studied about the production of biogas from various kinds of biomass. In Indonesia, there are many cows and lemongrass in nature. Therefore, this study is focused on the utilization of cow manure and lemongrass as the substrate for the production of biogas. The purpose of this study is to obtain the optimum condition of biogas production, including composition of the substrate and temperature of the bio digester. Besides, the addition of rumen bacteria was also investigated in this study. The composition of cow manure and lemongrass were varied at the ratio of 1:0, 3:1, 1:1, 1:3 and 0:1 in weight. The temperature of bio digester was also varied at room temperature, 39℃, and 50℃. Result shows that the volume of biogas produced by using lemongrass was less than using cow manure. However, the mixture of cow manure and lemongrass improved the quality and quantity of biogas produced. Based on the results, the highest volume of biogas at 890 mL and the highest percentage of methane at 14.90 % were produced from the composition of substrate with the ratio of 1:1 at the temperature of 50℃ with the addition of rumen bacteria. It was revealed that the higher temperature of bio digester was better for the production of biogas in the range of room temperature to 50℃. It was also proved that rumen bacteria are important for the production of biogas.