Microbial electrolysis cells (MECs) are considered as green technologies for H2 production with simultaneously wastewater treatment. Low H2 recovery and production rate are two key bottlenecks of MECs fed with real H2 fermentation effluent of biomass wastes. This work established a 1 L cylindrical single chamber MEC and enriched electroactive anodic biofilms from cow dung compost to overcome the bottlenecks. These MEC components (platinum-coated cylindrical titanium mesh cathode and cylindrical graphite felt anode) were arranged in a concentric configuration. A high H2 production rate of 6.26 ± 0.23 L L-1day-1 with H2 yield of 5.75 ± 0.16 L H2 L-1 fermentation effluent was achieved at 0.8V. The degradation of acetate and butyrate (main components of H2 fermentation effluent) could reach to 95.3 ± 2.1% and 78.4 ± 3.6%, respectively. The microbial community analysis for anode showed the abundance of Geobacter (22.6%), Syntrophomonas (8.7%), and Dysgonomonas (6.3%) which are responsible to complex substrate oxidation, electrical current generation, and H2 production. These results prove the feasibility of cylindrical single-chamber MEC for high H2 production rate and high acetate and butyrate removal from H2 fermentation effluent.