Soil microbial biomass and its stoichiometric ratios are key indicators of soil quality, driving soil nutrient cycling. Microbial Biomass Carbon (MBC), Microbial Biomass Nitrogen (MBN), Microbial Biomass Phosphorus (MBP) and its stoichiometric ratios is linked to slope aspects and tillage type-related changes, but little is regarded about how slope aspects and tillage types influence soil microbial biomass and its stoichiometric ratios in karst trough valley with severe rocky desertification and complex geological structures. Therefore, our study examined two slope aspects (sunny and shady slopes) and two tillage types (tillage lands and abandoned farmlands) to assess their impacts on soil microbial biomass and its stoichiometric ratios in karst trough valley. The results revealed that the Natural moisture Content (NC), Organic Carbon (OC), Total Nitrogen (TN), C:P, and N:P ratios were lower on sunny slope than on shady slope. Moreover, we observed that the MBC, MBP, and MBN:P ratios on sunny slope were significantly lower than on shady slope (4.87%, 17.09%, and 79.94%, respectively). Meanwhile, the MBC, MBC:P, and MBN:P ratios in abandoned farmlands were greater than those in tillage lands. Additionally, the results of the Pearson correlation analysis and path analysis indicated that the C:P and N:P ratios are pivotal factors influencing microbial biomass and its stoichiometric ratios. Overall, our research revealed that slope aspect and tillage type are key factors influencing the cyclic response of soil microbial biomass. This study provides valuable insights into the effective mitigation of rocky desertification and the enhancement of land quality in karst trough valley.