Display omitted •The effect of carbon-to-nitrogen ratios in mDON generation dynamics was considered.•The simulation of mDON generation in four-denitrification was accurate (R2 = 0.909–0.986).•mDON formed in real wastewater was provided a reasonable prediction (1.16–1.63 mg/L).•Production of new nitrogenous molecules confirmed the model applicability.•High carbon-to-nitrogen ratios are of benefit to reduce effluent-associated ecological risk. Quantifying microorganism-derived dissolved organic nitrogen (mDON) formed from microbial activities is a prerequisite for reducing effluent nitrogen. In this work, a new model based on activated sludge model for nitrogen (ASMN) is proposed for determining mDON formation together with its dynamics in post-denitrification under different carbon-to-nitrogen (C/N) ratios of 3, 4, 5, and 6. This model incorporates mDON as a separate component among four-step denitrification and introduces three new mDON-related parameters (i.e., fH,mDON, KH,mDON, and KI4SS) to develop mDON production and utilization equations. The model was calibrated and established with data from an experiment using synthetic wastewater, demonstrating its capability of accurately simulating mDON generation (R2 = 0.909–0.986). Accordingly, the simulated results successfully captured the experimental trends, suggesting that higher C/N ratios were preferred for mitigating mDON generation. The model applicability was further tested by predicting mDON generation in real wastewater, producing reasonable predicted results of formed mDON (1.16–1.63 mg/L). These predicted results were also indirectly confirmed by newly produced nitrogenous molecules (i.e., lipid-, carbohydrates-, and protein/amino sugars-like molecules). Overall, this work offers a tool to quantify mDON which benefits controlling mDON toward minimizing the adverse impacts of effluent organic nitrogen on receiving waters.