Mushroom cultivation waste (MCW) is a polypropylene bag stuffed with wood flour and nutrients for growing mushroom, which is a feasible feedstock for anaerobic biohydrogen production owing to its abundant availability, high organic and nutrient content. This study optimized the seed inoculum from various waste sludges (sewage sludge, cow dung and pig slurry), nutrient addition and operation conditions (moisture content and MCW powder particle size) for maximal biohydrogen production by solid-state fermentation (SSF). SSF batch test was operated at a MCW 3 g total volatile solid (TVS)/L, temperature 55°Cand rotation speed of 15 rpm with a vertical rotative shaker. The peak hydrogen production performance of hydrogen production rate (HPR) 9.50 mol H 2 /kg-d and hydrogen yield (HY) 0.29 mmol H 2 /g TVS) are obtained using sewage sludge 2 seed inoculum, nutrients addition, moisture content 70% and particle size of 1.190~0.590 mm. The results show that the MCW has the potential for hydrogen production by anaerobic mixed microflora using solid-state fermentation. The bioenergy of 1842 kWh while using SSF to conver MCW to produce biohydrogen and it could reduce CO 2 emission of 114-178 kg per year comparing using fossil fuel such as coal, fuel oil and natural gas.