Abstract: To elucidate the contribution of soil microbial necromass to organic carbon accumulation under carbon and nitrogen inputs, this study used a microcosm experiment to explore the effect of nitrogen fertilizer combined with carbon dots on the content of microbial necromass carbon in fluvo-aquic soils. Carbon dots (CDs) is one of the nano-carbon materials deriving from organic waste matters of salvia miltiorrhiza. Four treatments were set up: control, neither N nor CDs addition (CK), CDs amendment (CDs, w=0.15%), N fertilizer addition (N, urea), N fertilizer and CDs amendment (N+CDs). Each treatment had three replicates for incubation period. The results show that compared to CK treatment, the content of inorganic nitrogen (SIN) under CDs treatment significantly decreased by 29.1% (P < 0.05). Compared with N treatment, the SIN content of N+CDs treatment significantly decreased by 32.8%, simultaneously, soil urease and nitrite reductase activities also decreased. The soil dissolved organic carbon (DOC) contents not only increased in CDs treatments, but also the functional group structure of DOC was altered by CDs treatment compared to CK treatment. Compared to N treatment, the content of DOC significantly increased by 28.8% under N+CDs. It also increased the contents of amines and aromatic compounds in DOC. Compared with N treatment, the contents of bacterial necromass carbon (BNC) and fungal necromass carbon (FNC) in N+CDs treatment significantly increased by 29.5% and 17.7% (P < 0.05), respectively. Meanwhile, they also increased the relative contribution of the accumulation of organic carbon from soil microbial necromass. Regression analysis revealed a correlation between total bacterial and fungal necromass carbon and soil physicochemical properties. Further analysis through establishing structural equation modeling reveals that aromatic compounds in DOC were the main factors to regulate BNC, while FNC was primarily regulated by the net nitrification rate and the average molecular weight of DOC. In this study, nitrogen fertilization combined with CDs had primarily driven microbial necromass carbon accumulation by altering the functional group structure of dissolved organic carbon and inorganic nitrogen content.