This study aimed to evaluate the environmental impact of 1000 kg h−1 wheat straw to produce biofuel via fast pyrolysis with three different hydrogen production processes by the life cycle assessment (LCA) based on Chinese Life Cycle Database (CLCD). The primary energy depletion (PED), global warming potential (GWP), abiotic depletion potential (ADP) and respiratory inorganics (RI) impact categories of 1 MJ biofuel produced were employed for comparison. In case 1, the hydrogen was derived from natural gas steam reforming, and all the bio-oil was hydrotreated to produce the biofuel. In case 2, a part of the aqueous phase was reformed to produce hydrogen, whereas the remaining bio-oil was hydrotreated to produce biofuel. In case 3, all the aqueous phase of bio-oil was reformed to produce hydrogen, a part of hydrogen generated by reforming was used to oil phase hydrotreated and the excess hydrogen was considered as a co-product. Our results show that the PED, GWP, ADP and RI of case 3 are 0.1355 MJ, −17.96 g CO2eq., 0.0338 g antimonyeq and 0.0461 g PM2.5eq.. Compared with conventional diesel, the PED, GWP, ADP and RI of case 3 were reduced by 89.81, 117.44, 1.74 and 85.03%, respectively. The results of sub-process contribution analysis and sensitivity analysis suggested that the electricity consumption for the bio-oil production has the maximal effect on the total PED, GWP and RI of case 3, whereas the amount of fertilizers in the biomass production sub-process has the maximal effect on the total ADP.