Abstract: Intensive tillage has strongly altered the physical structure and biochemical patterns of Mollisols in Northeast China. There is a serious threat to the sustainable productivity of the cropland. However, it remains unclear on the changing pattern of the Mollisols quality and its key influencing factors under long-term cultivation. In this study, the upland fields of Mollisols were selected in the Sanjiang Plain with a series of cultivation years (5, 20, 30, 60, 85, and 100 years). A systematic analysis was also made on their physical, chemical, and microbial indicators. An optimal data set was established to assess the Soil Quality Index (SQI). Furthermore, the Random Forest Model was used to combine with the literature synthesis. The key drivers were determined in the soil quality under long-term cultivation. The results showed that the soil organic matter (SOM) content exponentially decreased with increasing cultivation years. In particular, the SOM content decreased by 49.9% from 42.3 to 21.2 g/kg during the initial 30 years, while the decrement was 12.6% during the period of 60‒100 years. Dissolved organic matter (DOM) showed that there was a similar but faster loss rate than SOM, with a decrement of 56.1% after 100 years of cultivation. There was a significant decrease in the soil pH, total nitrogen, total phosphorus, mean weight diameter of aggregates (MWD), and microbial biodiversity index; On the contrary, the upward trends were observed in the total potassium, bulk density, and microbial biomass carbon. The SQI was composed of the total nitrogen, bulk density, and the multi-shannon index of the bacterial and fungal communities across the trophic levels. There was a linear decrease with the increasing cultivation year. Random forest modeling showed that the MWD and SOM were the key factors with the highest explanations for the variations of SQI. Structural equation modeling showed that the cultivation mainly reduced the soil quality. The aggregate structure was destroyed to reduce the SOM and DOM content. Furthermore, the magnitude and rate of the SOM decline in the Sanjiang Plain were significantly higher than those of the average of the Mollisols region of northeast China, which was 1.5 times higher than that of the Songnen Plain at a 100-year cultivation scale. There were higher initial SOM contents before reclamation and more drastic reclamation perturbations in the Sanjiang Plain. In conclusion, the fragmentation of soil aggregates and low SOM caused by long-term cultivation were the main triggers of the continuous degradation of the soil quality. The findings can also provide an important theoretical basis for the soil quality and sustainable utilization of the Mollisols cropland in the Sanjiang Plain.