In situ investigation of the affect of pretreatment lignin distribution and interplay with enzymes can present perception into the mechanism of lignin limiting the enzymatic effectivity of cellulose. One of many key focuses of this paper is to elucidate the variations in cellulase hydrolysis attributable to floor lignin after pretreatment (together with pseudo-lignin, deposited on biomass floor lignin and soluble lignin) in comparison with residual lignin within the pretreated materials. Notably, the mixture of various non-destructive spectroscopies (atomic drive microscopy (AFM), floor plasmon resonance (SPR), and fluorescence spectrometry (FLS)) have been used to look at the non-productive adsorption mechanisms of cellulase with differing lignin fractions from pretreated poplar. The outcomes confirmed that floor lignin (SL) from pretreated poplar had virtually no inhibitory impact on Avicel hydrolysis yield. In distinction, residual lignin (RL) in pretreated poplar considerably inhibited Avicel hydrolysis yield from 74.6% to 50.94%. Characterization outcomes revealed that SL has decrease hydrophobicity, molecular weight, and linkage content material (β-O-4, β-β and β-5), which is considerably completely different from the construction of RL. The multi-dimensional non-destructive spectroscopy indicated that the interplay forces between SL and cellulase fractions (0.044-2.04 nN) have been smaller than that of RL and cellulase fractions (0.11-3.62 nN). The SL binds to cellulase primarily by way of hydrogen bonds and van der Waals forces, whereas the RL interacts primarily by way of hydrophobic forces. Moreover, SL sure to cellulase is extra prone to dissociate in contrast with RL. This work gives a basic understanding of lignin-cellulase interactions for successfully changing lignocellulose into biofuels.