To investigate the impact of different H2O2 concentrations on the Fenton-like systems of H2O2/biochar, this study examined the mechanism of the physical structure and environmental persistent free radicals (EPFRs) of biochar during diethyl phthalate (DEP) removal by the Fenton-like system. The peak-splitting method was utilized to differentiate EPFRs types in cotton stalk biochar produced at different temperatures. High-temperature environments promote π-electron delocalization, which facilitates phenyl π free radicals and σ-π oxygen-containing free radicals. By analyzing relationships between the removal rate K1 and removal constant Kobs of DEP with the structural properties of biochar, it was discovered that EPFRs concentrations in biochar had a significant positive correlation with K1 (r = 0.92) and Kobs (r = 0.97). Different H2O2 concentrations added to the biochar removal system resulted in varied DEP removal efficiency. Among them, CS500, CS550, and CS600 exhibited superior DEP removal efficiency when H2O2 concentration was 5 mM.