To explore the underlying reasons for the decreased enzymatic hydrolysis yields of the acid pretreated softwood under the higher pretreatment severity, the residual bulk lignins, as one type lignin fraction with stronger inhibition, were isolated from larch (softwood) pretreated with different pretreatment severity in this study. The lignin structure changes during the pretreatment were comprehensively investigated by nuclear magnetic resonance (NMR) analysis. The larch lignins addition decreased the enzymatic hydrolysis of Avicel from 68.43% to 25.9–39.3%. The pretreatment intensified the inhibition of isolated lignins on enzymatic hydrolysis of Avicel. Furthermore, quartz crystal microbalance (QCM) analysis was applied to investigate the interaction between enzyme and lignins, distinguishing the reversible and irreversible enzyme adsorption. It demonstrated that the obviously higher irreversible adsorption of enzyme was observed on lignins from pretreated substrates with the higher severity. Additionally, the correlation between structure and inhibition of residual lignins in the enzymatic hydrolysis was then investigated with Pearson’s correlation heatmap. The findings can be used to develop theoretical guidelines for alleviating the residual lignin inhibition in the enzymatic hydrolysis of acidic pretreated softwood.