Biomass pretreatment is often evaluated based on the degree of delignification and the yield of enzymatic hydrolysis. Thus, the phase delignification and enzyme diffusion kinetics models were utilized to describe the deconstruction mechanism of wheat straw treated with two-step radical oxidation. The oxidation started with a superoxide radical anion, followed by a second radical treatment that synergizes the advantages of both radical systems. Two-step superoxide radical anion and two-step superoxide-hydroxyl radical pretreatments were the most effective pretreatment based on the degree of lignin removal and sugar release. These treatments resulted in 65 and 46% decrease in lignin content and 85.75 and 73.96% increase in glucan recovery, respectively. The delignification kinetics constants, k(1), k(2), and k(3), were found to be 1.950, 1.085, and 1.085 min(-1), respectively, for two-step pretreatment with superoxide radicals and 1.094, 0.717, and 0.717 min(-1) with superoxide-hydroxyl radical pretreatment, whereas the kinetics constants were 0.485, 0.390, and 0.390 min(-1) for the control. The structural diffusion resistance constant, n, was 0.205-0.359 for the control and 0.185-0.305 for the one-step superoxide treatment, 0.114-0.304 for two-step superoxide treatment, and 0.179-0.249 for two-step superoxide-hydroxyl radical treatment. These results justify that high lignin removal does not always contribute to a better enzymatic hydrolysis rate, likely because oxidized lignin products on the surface of biomass can restrict enzyme accessibility.