Co-pretreatment of wheat straw by urea and microorganism was conducted. The effects of supplement dose of microorganism in biological pretreatment of wheat straw on its anaerobic digestion for biogas production were investigated by using a self-made anaerobic digestion device with pretreated wheat straw as raw materials. Results showed that the total yield of biogas reached the maximum of 7968mL, which was 23.11% higher than the control, when the urea concentration, pH value of biological pretreatment and ratio of Phanerochaete chrysosporium to Trichoderma reesei added into the wheat straw were 35g/L, 4, and 1∶1, respectively. The values of VFA, CH4 and pH value were located in normal range. The maximum CH4 content was 51.33%, which was 6.01% higher than that of control. The fermentation time was shortened to 23d during the whole experiment.