In our work, thermal behavior and kinetic characteristics of spent lark mushroom substrate were evaluated to elaborate the thermal decomposition mechanisms and explore the influence of heating rate by using thermogravimetric analyzer and Coats–Redfern method. The study of pyrolysis temperature of raw material was also operated at the range of 410–530 °C, under the feeding rate 0.36 g/min, and the nitrogen flow 16 L/h. The results showed that the maximum bio-oil yield was obtained at 470 °C with the yield of 14.4 wt.%. The analysis of Fourier transform infrared spectrometer and gas chromatography coupled with mass selective detector indicated that the target liquid production was consisted of phenols, hydrocarbons and other components. Simultaneously, the low oxygen and high hydrogen content in bio-oil was also determined by elemental analysis. Based on the above-mentioned results, we demonstrated that the bio-oil obtained from the substrate had high utilization value as a potential energy resource.