Biomass pyrolysis technology has important developmental prospects for biofuels and chemicals. Biochar as one of main pyrolysis products has excellent performance in soil improvement and adsorption of harmful elements. The environmentally persistent free radicals (EPFRs) in corn stalk powder and biochar obtained by pyrolysis at different temperatures were tested by electronic paramagnetic resonance spectrometry. After pyrolysis treatment, the biochar had a large number of stable free radicals. With increased pyrolysis temperature, the peak width of the free radicals signal in biochar decreased remarkably, and the widest peak of free radicals signal in biochar was presented at 450 °C, which was 0.69×10-4 T. The g-factor of free radicals in biochar decreased continuously as the temperature increased, while the peak height of free radicals first increased and then decreased. The peak height at 600 °C was only 23.8% of the peak height at 500 °C. The concentration of EPFRs in biochar increased with the increase of temperature from 450 °C to 500 °C, while it decreased at higher temperature (>500 °C). This phenomenon was similar to the trend of bio-oil collection efficiency. The experimental results showed a correlation between EPFRs of biochar and bio-oil yield at different pyrolysis temperatures.