Long-term frequent tillage would cause black soil degradation and serious soil erosion as soil microbial communities and soil structure are extremely sensitive to tillage process. However, there is no unified conclusion on the relationship between the distribution of soil water-stable aggregates (WSAs), and microbial community construction and diversity under long-term tillage in black soil during different seasons. In this study, we used wet-sieving method to evaluate the composition and stability of soil WSAs and employed Illumina MiSeq high-throughput sequencing technology to study the diversity, taxonomic composition and co-occurrence network properties of microbial community, comparing outcomes between uncultivated soil and long-term cultivated soil for 60 years in Keshan farm of Heilongjiang Province. The results showed that after long-term tillage, the proportion of larger than 1 mm WSAs reduced by 34.17–51.37%, and the stability of WSAs, soil pH, organic matter (OM), total nitrogen (TN) contents decreased significantly in all seasons (P < 0.05), while soil available phosphorus (AP) and available potassium (AK) contents increased remarkably (P < 0.05). The diversity of bacteria increased, while that of fungi decreased. Soil fungal communities were more susceptible to long-term tillage than bacterial and archaeal communities. Actinobacteria mainly exist in large WSAs (˃1 mm), and when their relative abundance is high, it is beneficial to improve the water-stability of black soil; while Proteobacteria and Gemmatimonadetes may exist in small WSAs (˂1 mm), whose high relative abundance will weaken the water-stability of black soil. The experimental results provide a scientific theoretical basis for sustainable utilization of black soil.