In resent years, the phenomenon that China’s eastern coast was invaded by Enteromorpha prolifera because environmental change and anthropogenic factors occurred frequently. The significance of the study on fermentation of E. prolifera is in order to make the high quality macroalgae biomass feedstock to be used rationally and efficiently, and to turn waste into treasure; On the other hand, it is a method to enrich types of raw materials of biogas in fermentation process to achieve the purpose of the development of renewable energy. In this paper, we research the effects on the biomass production by anaerobic fermentation of E. prolifera of different granule sizes(coarse particles 0.30-0.85 mm, medium grained 0.15-0.30 mm, fine grained 0.075－0.15 mm) and under different pretreatment methods(thermal pretreatment, dilute alkali pretreatment, acid pretreatment). The results show that, biogas production rate of volatile solids (VS) in anaerobic fermentation process of using E. prolifera of different granule sizes mentioned above are 386.9 mL/g, 434.4 mL/g and 436.5 mL/g.The production of biogas of E. prolifera at medium grained showed 12.26% improvement than that at coarse particles. The experiments of E. prolifera at fine grained showed 0.49% improvement than that at medium grained. It shows that the smaller the particle size, the more improvement of utilization of E. prolifera in biogas production. It was found that, compared with the group without pretreatment, efficiency of biogas production of the three pretreated groups (thermal, dilute alkali and acid pretreatment methods) showed 13.13%, 11.39% and 3.63% improvement when all the groups were under the same conditions(the same as the previous experiment). Alkaline pretreatment method showed the best result, biogas production rate of volatile solids of this group is 493.3 mL/g. Alkaline pretreatment method improved biogas production efficiency in fermentation effectively, and the pH value in the process is relatively stable. This study tries to provide a theoretical reference to the research of biogas production process by anaerobic fermentation of E. prolifera.