In resent years, the phenomenon of China's eastern coastal was invaded by Enteromorpha prolifera because of environmental change and anthropogenic factors occured 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 turning waste into treasure; On the other hand, It is a method to rich types of raw materials of biogas in fermentation process to achieve the purpose of the development of renewable energy. In this paper, We research in the effects on the biomass production by anaerobic fermentation of E. prolifera at different granule sizes(coarse particles 0.30-0.85mm, medium-grained 0.15-0.30mm, fine-grained 0.075-0.15mm) 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 at different granule sizes witch was mentioned above are 386.9ml/g, 434.4ml/g and 436.5ml/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 493.3ml/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.