Enzymatic hydrolysis is a method to generate biofuel from biomass, and autohydrolysis is a popular method to pretreat biomass prior to enzymatic hydrolysis. The primary aim of the present study was to determine the role of lignin produced in the autohydrolysis process on the enzymatic hydrolysis of biomass. The HSQC and ³¹P NMR analyses confirmed that β-O-4 of lignin was reduced, while β-5, β-β, and S/G-ratio of lignin were increased with intensifying the hydrolysis intensity. The increase in the hydrolysis intensity significantly enhanced the condensed and non-condensed phenolic OH group of lignin. Interestingly, the cellulase enzyme adsorbed more on lignin that had more phenolic content, and its association with lignin reduced its activity for hydrolyzing cellulose microcrystals. Strong negative correlations were observed between the enzymatic hydrolysis yield and the condensed S-OH (r² = 0.978) and G-OH (r² = 0.961) of lignin generated in the autohydrolysis process.