Structural optimization of activated carbon (AC) mainly relies on experience, which depends on the intrinsic structure of biochar, processing conditions, and the interplay of both parties. A fundamental understanding of the pore structure evolution related to the intrinsic structure and composition remains a challenge. In this work, spent mushroom substrate, a rapidly growing byproduct of the mushroom cultivation industry, is used as model biomass to prepare AC under CO2 activation. The structure and composition of the AC products with different activation durations were systematically analyzed with several characterization techniques including N2 adsorption–desorption, scanning electron microscopy, X-ray diffraction, and Raman spectroscopy. A multipeak separation method is developed that enables quantitative analysis of carbonized lignin and carbonized cellulose. A peak area ratio parameter is proposed to describe the retention of cellulose. It is revealed that higher retention of carbonized cellulose corresponds to a larger Brunauer–Emmett–Teller (BET) surface area, demonstrating the dominant role of cellulose in the pore structure development process. This work not only provides a qualitative correlation between cellulose and rich porous structure but also offers a new quantitative tool to understand the structure–composition relationship during the pore evolution process.