Abstract: Nitrate pollution, commonly originating from agricultural wastewater, livestock breeding, and aquaculture, poses signifiant environmental challenges. Heterotrophic denitrification represents an effective method for nitrate removal, with the selection of an appropriate organic carbon source being critical to its success. Woody biomass, commonly employed as the carbon source in denitrification, suffers from slow denitrification rates and significant residual organic matter accumulation owing to muti-factor influences including the lignin content, pH, and reaction temperature. In this study, multi-factor batch experiments using collected woodchips were systematically designed via response surface methodology (RSM) to simulate and comprehensively evaluate the denitrification process, which was divided into distinct carbon release and denitrification phases. The main compositions and spectral characteristics of residual organic matter generated during the process were identified using advanced analytical techniques. Results show that the carbon release phase was affected by temperature, lignin content, and pH, in descending order of impact. The interactive effects of temperature and lignin content are more significant in regulating key process metrics, including DOC release rate, DOC utilization efficiency, and TN removal rate. Conversely, the combined effect of pH and lignin content exerts a greater control over the stoichiometric outcome, namely the C/N ratio. Nevertheless, the relative influence of these factors varied during the denitrification phase. Notably, low pH conditions significantly restricted the carbon release rate for low-lignin woodchips but did not markedly impacted the denitrification rate, resulting in reduced organic residue. Specifically, dissolved organic carbon (DOC) release increased significantly with rising temperature and decreasing lignin content, with DOC release rates for low-lignin woodchips being 2.7 to 3.8 times higher than natural woodchips. The DOC utilization efficiency improved from 26.5% to 50.4% as lignin content decreased. The total nitrogen removal rates increased by 4.9 to 6.1 times for low-lignin woodchips compared to natural woodchips. The ratio of consumed DOC to total nitrogen removal dropped from 1.600 to 0.868, reflecting optimized carbon-nitrogen coupling. Low residual DOC levels were consistently observed under mildly acidic conditions throughout the experimental period. The coefficient of determination R² and the adjusted R² of each model are both greater than 0.90, while the predicted R² values are all greater than 0.71, and the difference between the adjusted R² and the predicted R² is less than 0.2. This indicates that the fitted model can be used to predict the changes in response values under different factor conditions. The Total Nitrogen (TN) removal rate increases significantly as the lignin content decreases. The TN removal rate of NW is 0.082 mg/(g·d), whereas LM1 and LM2 reach 4.9 and 6.1 times that of NW, respectively. Based on maximizing DOC release rate, DOC utilization rate, and TN removal rate as the optimization criteria, the optimal conditions were determined to be a temperature of 34.9  ℃, a pH of 6.9, and a lignin content of 17.6%. Under optimized parameter conditions, the DOC release rate, DOC utilization rate, and TN removal rate reached maximum values of 0.321 mg/(g·d), 58.1%, and 0.593 mg/(g·d), respectively, with the maximum deviation from the model-predicted values not exceeding 2.0%. Under these conditions, both the carbon release and denitrification capacity of the woody carbon sources are significantly enhanced. Woodchips offer a sustainable option by reutilizing woody waste from wood processing or tree harvesting. Their lignin content could be adjusted through changing chemical treatment to meet diverse nitrate removal requirements. Therefore, this study results contribute valuable insights for wood waste recycling and nitrate contamination control in the agricultural practices.