To understand the effect of sulfuric acid-based phenoxyethanol pretreatment on the enzymatic digestibility of bamboo, the changes of lignin/xylan content and their intercellular structures in pretreated bamboo were investigated by different spectrum technologies. The maximum removals of lignin and xylan achieved by pretreatment with phenoxyethanol-acid (1%, w/v) ratio of 4:1 at 120 °C resulted in pretreated bamboo with an enzymatic digestibility of 79.25%. Micro-computed tomography revealed that the macrostructure of bamboo cell wall was destroyed, exposing the macrofibers. Based on the fluorescence intensity of lignin and antibody-labeled xylan, measured by fluorescence microscopy, most of the lignin was removed from the intercellular complex layer in thin-walled cells, and the destruction of main chain of hemicellulose was more severe than that of branched chains during the pretreatment process. In addition, the number of sub-structures, functional groups, and molecular weights of dissolved lignin in the pretreatment hydrolysate was closely related to the phenoxyethanol-acid ratio employed during pretreatment. Compared with the control group, the structures of dissolved lignin changed more significantly as the percentage of phenoxyethanol in the system increased. When the proportion of phenoxyethanol in the system increased, the number of substructures (β-O-4, β-5, and β-β) and functional groups showed a decreasing trend.