Abstract: Facing the dual challenges of the sustainable disposal of harvested cyanobacteria from eutrophic water bodies and the lack of bioavailable carbon for denitrification in sewage treatment plants with a low C/N ratio influent, this study aimed to develop and evaluate an integrated process for converting cyanobacterial biomass into an efficient liquid carbon source, thereby providing a synergistic solution for both waste valorization and enhanced nitrogen removal. To achieve this, cyanobacteria collected from Taihu Lake in China were first subjected to thermo-alkaline pretreatment to disrupt the cell walls. This was followed by mesophilic anaerobic fermentation to produce a volatile fatty acid (VFA)-rich fermentation broth. Due to the high ammonium nitrogen content, the broth was subsequently treated via vacuum thermal stripping, and the effects of pH, temperature, and stripping time on ammonia removal were systematically optimized. Finally, the denitrification performance of the de-ammoniated broth was assessed in both batch experiments and a continuous-flow reactor with activated sludge. The influences of the C/N ratio and pH were investigated, and the broth was compared with sodium acetate and glucose. Nitrogen species were monitored, and the denitrification potential (P_DN) was calculated. The broth produced by anaerobic fermentation contained 15,582.00 mg/L of VFAs, with acetate accounting for 72.94%. The optimized vacuum thermal stripping process achieved 93.77% NH₄ ⁺-N removal at pH 10.5, 53 °C, and 40 min, increasing the C/N ratio from 11.25 to 70.59, thus making the broth a suitable carbon source. Denitrification tests revealed that a C/N ratio of 6 and a pH of 7 were optimal, achieving 99.46% nitrate removal. Notably, the maximum nitrite accumulation was significantly lower than that observed in the sodium acetate system. The P_DN value for the cyanobacteria broth (0.192 g N/g COD) exceeded that of sodium acetate (0.176 g N/g COD) and glucose (0.150 g N/g COD). Operation of the continuous-flow reactor indicated that when the carbon source in the fermentation broth had a C/N ratio ≥ 5, the average nitrate removal efficiency remained stable at over 98.61%, and the average total nitrogen removal efficiency exceeded 82%, demonstrating stable and efficient denitrification performance. In conclusion, this study demonstrated that cyanobacterial biomass can be efficiently converted into a promising external carbon source for sewage denitrification through a sequential process comprising thermo-alkaline pretreatment, anaerobic fermentation, and vacuum thermal stripping. The fermentation broth exhibited better denitrification performance than commercial carbon sources like glucose and showed an advantage over sodium acetate in terms of lower nitrite accumulation and higher intrinsic carbon efficiency (P_DN). This verifies the technical feasibility and effectiveness of the proposed strategy, offering a sustainable solution for both algal waste mitigation and enhanced nitrogen removal in sewage treatment. Moreover, this circular strategy transforms environmental waste into a valuable resource, potentially reducing the carbon footprint associated with both algal disposal and synthetic carbon production. The complex components in the broth appeared to promote more stable denitrification kinetics with reduced accumulation of intermediates compared to pure compounds, suggesting operational benefits for improved process stability.