To assess the possible effects of lignin on enzymatic hydrolysis, the structural characteristics of milled wood lignin (MWL) isolated from reed straw and corn stover pretreated with liquid hot water (LHW) at different severities were investigated. The changes in the chemical structure of the MWL were characterized by gel permeation chromatography, elemental analysis, differential scanning calorimetry, thermogravimetric analysis, Fourier transform infrared spectroscopy, and 1H and 13C nuclear magnetic resonance. The results showed that the MWL isolated from reed straw and corn stover pretreated with LHW was more condensed. The non-conjugated and conjugated ketone groups, as well as the ether bonds of the MWL, disappeared after LHW pretreatment, while the phenolic hydroxyl and methoxy groups increased. The carboxyl groups of MWL from reed straw increased after LHW pretreatment, whereas the carboxyl groups of MWL from corn stover decreased. When MWL was added into the enzymatic hydrolysis system, the filter paper activity of cellulase, the protein content, and the conversion of cellulose to glucose all decreased.