Long-term fertilization is considered as the most effective practice in organic C accrual in intensively managed paddy soils. However, the contribution and mechanism of microbial necromass C for soil organic C accrual, and the effective incremental component in paddy soils under the different fertilization remain unclear. To address the knowledge gap, we collected soils from a 31-year long-term fertilization paddy field including no fertilization (control), mineral fertilizers alone (NPK), mineral fertilizers plus rice straw incorporation (NPK + S), and mineral fertilizers plus manure incorporation (NPK + M). Living microbial biomass and their necromass were quantified by the biomarker analysis of phospholipid fatty acids and amino sugars, respectively. Results showed that the three fertilization treatments significantly increased the bacterial and fungal biomass in both 0–10 and 10–20 cm layers, which were universally greatest in NPK + M. The microbial necromass C was little affected by 31-year mineral fertilization; however, mineral combined organic fertilizers incorporation greatly stimulated microbial necromass C accumulation. Besides, soil treated with NPK + M showed 3.55 ± 0.12 g kg−1 higher accumulation of microbial necromass C than NPK + S, because manure application increased the abundance of Gram-positive r-strategy bacteria (mainly Firmicutes and Actinobacteria), and enriched filamentous fungi (Ascomycota). Compared to the control, the increase in the microbial necromass C contribution to SOC was lowest in NPK and highest in NPK + M, mainly derived from the variation in the contribution of bacterial necromass. Consequently, this study suggested long-term mineral combined manure application in subtropical paddy fields could effectively increase soil C sequestration by strengthening microbial necromass accumulation. Bacterial and fungal necromass C accrual was closely associated with living microbial biomass and their community structure. Given the C sequestration potential of manure in paddy soil, as the crucial resource of livestock and poultry waste in this region, it should be incorporated into subtropical agricultural ecosystems.