Abstract: Vinegar residue (VR) is one of the primary byproducts in Chinese vinegar production. It is rich in the lignocellulosic resources. However, its efficient resource utilization has been hampered by the recalcitrant structure of the lignin-carbohydrate complex. In this study, VR resource utilization was established using ammonia pretreatment, enzymatic saccharification, and microbial conversion. The yield of VR VR-reducing sugar was also enhanced after treatment. The hydrolysate was utilized for the single-cell protein (SCP) production. Firstly, single-factor experiments were conducted to determine the suitable ranges of the key parameters for the ammonia pretreatment (ammonia loading, temperature, time, and moisture content). Secondly, a Box-Behnken response surface method (RSM) with the 17 trials was employed to optimize the pretreatment parameters, in order to maximize the enzymatic reducing sugar yield. Pretreated samples were hydrolyzed using Cellic CTec3 HS cellulase. Three yeast strains were evaluated (Saccharomyces cerevisiae 2, Candida tropicalis GB3, and Meyerozyma caribbica GD1) for their capacity. The optimal SCP producer was identified to utilize the reducing sugars in the VR hydrolysate (VRH). Taking the VRH as the basal medium, the a systematic investigation was made to explore the effect of the nitrogen source combination (aqueous ammonia and yeast extract), growth factors (MgSO₄, ZnSO₄, MnSO₄, biotin, pantothenic acid, and thiamine), and carbon-to-nitrogen ratio (C/N 5:1-20:1) on the biomass production. Finally, the high-yield SCP medium parameters were obtained to validate in shake-flask and high-cell-density fermentations. Results indicated that the ammonia water dosage, temperature, time, and moisture content were all significantly impacted the saccharification efficiency (P<0.05). The optimal conditions of ammonia pretreatment were 21% w/w NH₃·H₂O, 64 °C, 62 h, and 30% moisture content. The reducing sugar yield reached 252.89 mg/g VR, indicating a 123.6% increase, compared with the untreated control (113.98 mg/g) (P<0.05). Among the three yeast strains, C. tropicalis GB3 demonstrated the superior performance. Moreover, 60.68% of the VRH reducing sugars were utilized to achieve an initial dry cell weight (DCW) of 5.36 g/L, which was significantly higher than the rest (P<0.05). Biomass production was significantly enhanced to employ a 3:2 ammonia-yeast extract nitrogen source. The MgSO₄ was then added to optimize the C/N ratio to 10:1. The optimal high-yield SCP process was comprised: VRH (containing 31.97 g/L reducing sugars, and 12.45 g/L crude protein) 1 L, supplemented with 8.3 g/L yeast extract, 10.4 g/L glucose, and 2 g/L MgSO₄, with an initial pH of 6.5. After 48 h of shake-flask fermentation, the DCW and dry protein weight reached 17.39 g/L and 7.83 /L, respectively. These typical increases of 131.00% and 151.61% were achieved he optimal combination, compared with the control using VRH medium (7.53 g/L DCW and 3.11 g/L protein) (P<0.05). Subsequently, the high-cell-density fermentation in a 5-L bioreactor for 24 h was further elevated the DCW and protein yield to 24.64 g/L and 11.40 g/L, respectively, corresponding to increases of 61.79% and 38.35%, respectively, compared with the growth in YPD medium (P<0.05). While the VRH fermentation was reduced the reliance on conventional carbon sources, compared with the YPD. The functional composition of the resulting biomass was reduced after fermentation. Crude protein content decreased by 14.50%, mannan content by 18.78%, nucleotide content by 49.53%, essential amino acid content by 16.06%, and total amino acid content by 9.99% (P<0.05). This reduction was likely attributed to the VRH lacking a complete amino acid profile, nucleic acid precursors, and B vitamins. The high C/N ratio of the optimal medium was coupled for with the metabolic resources being preferentially allocated towards the biomass synthesis over functional component accumulation. The cost reduction was attributed to the inorganic nitrogen that provided by residual ammonia in VRH. Ammonia pretreatment was effectively improved the cellulose accessibility. Efficient enzymatic saccharification and microbial conversion were realized using the optimal system. The VRH can be expected to serve as a viable low-cost substrate for the SCP production. The C. tropicalis GB3 was achieved the high biomass and protein yields. This finding can also provide for the industrial-scale valorization of the lignocellulosic waste. There was the synergy between resource recovery and sustainable protein production. Future research should focus on the functional protein quality using precursor supplementation and metabolic engineering.