Understanding the interactions between lignin and cellulase will help better design biomass pretreatment and enzymatic hydrolysis processes. Ethanol organosolv lignins (EOLs) were isolated from organosolv pretreatment of sweetgum (SG) and loblolly pine (LP) under different temperatures. The structural changes of EOLs were determined by C-13 NMR and heteronuclear multiple bond correlation NMR to elucidate their positive and/or negative effects on enzymatic hydrolysis. Alkylation of lignin with ethanol resulting in the etherification of C-alpha was observed in both EOL-SG and EOL-LP. The effect of EOL-SG on enzymatic hydrolysis was correlated with the content of ethoxyl groups. The lignin alkylation in EOLs appeared to be reversible at high temperature, resulting in the inverse change of their effects on enzymatic hydrolysis. A strong correlation was observed between the binding affinity of cellulases onto EOLs and their corresponding hydrolysis yields. Lignin alkylation likely reduced the affinity of enzyme onto lignin, which can enhance the enzymatic hydrolysis.