Excessive use of fossil fuels has led the world to severe energy crisis and environmental pollution. Biogas has been considered as one of the substitute to conventional fossil fuels because of its high energy density and clean combustion products. Fermentative biogas production with algae biomass as the substrate has become a topic of interest for many researchers and scientists. In this research, the fermentation of pre-treated green algae (Enteromorpha) and sludge is carried out to explore the effect of microwave pre-treatment conditions on biogas production. Response Surface Methodology (RSM) is used to optimize the fermentation conditions for the microwave pre-treatment by considering the pre-treatment microwave power, time and liquid-solid ratio. Results showed that the microwave power of the microwave pre-treatment has the most significant effect on the fermentation of the Enteromorpha. The optimum conditions have been achieved at microwave power of 656.92 W, pre-treatment time of 5.10 min and liquid-solid ratio 33.63:1. From these optimum conditions, it was found that microwave pretreatment power about 600 W had a better effect. It was necessary to have the anaerobic fermentation experiment with this condition to observe the real fermentation effect. Optimum conditions provide highest amount of COD and reducing sugar increase of 10420 mg/L and 0.77-0.79 g/L respectively. The increase in COD and reducing sugar shows that the pretreatment has improved influence over fermentation. The peak biogas production amount of microwave pre-treated 20:1, 6 min group reached 244 mL, however the control group only reached 188 mL in total. The study will help in microwave pretreatment with anaerobic algae (especially Enteromorpha) fermentation.