Long-term cultivation tends to cause thinning and even partial disappearance of Mollisols, which largely limits the sustainable development of agriculture. Cultivation duration greatly affects soil pore structure and biochemical properties of Mollisols. This study aims to elucidate the response of aggregate pore structure, stability, and biochemical properties of soil during degradation under long-term cultivation in Mollisols. The study involved four croplands with different cultivation durations (20, 40, 60, and 100 years; designated as AL20, AL40, AL60, and AL100, respectively) and one undisturbed forest land (FR). Samples were collected from three soil depths (0–20, 20–40, and 40–60 cm). Pore structure and biochemical properties were analyzed by X-ray Computed Tomography (CT) and standard method. As a result, the aggregate stability and biochemical properties of soil showed different degrees of degradation with the extension of cultivation duration. The mean weight diameter, soil organic carbon, total nitrogen, and microbial biomass carbon followed the order of AL20 > FR > AL40 > AL60 > AL100, while the soil bulk density exhibited an opposite trend. FR and AL20 showed significant differences from other sites (P < 0.05). The CT results revealed that total porosity and pore number approximately decreased with cultivation duration, with their maximum appearing in FR and minimum occurring in AL100. The pore shape and pore size of aggregates were dominated by elongated pores (> 65%) and porosity > 100 µm, respectively, both of which declined with increasing cultivation duration. Similarly, the fractal dimension and connectivity were the highest in FR. These results demonstrated that the pore structure of soil aggregates tended to be "simplified" with increasing cultivation duration. Soil structure index, which varied significantly with cultivation duration, was used to comprehensively evaluate the soil structure. The results provide important insights into soil structural degradation caused by long-term cultivation.