Global protein shortages and increasing demand for sustainable animal feed necessitate alternative protein sources. Single-cell protein (SCP) derived from agricultural waste through microbial fermentation represents a promising circular bioeconomy solution. This study investigates an integrated strategy combining optimised acid pretreatment and yeast co-culture fermentation to enhance SCP yield and protein content from vegetable-based agro-waste substrates. A multi-phase experimental approach was implemented, beginning with screening of monoculture and co-culture yeast systems using Saccharomyces cerevisiae, Candida tropicalis, and Candida krusei. Co-culture fermentation significantly outperformed monocultures, with the S. cerevisiae, C. krusei consortium producing 4.58 g/L biomass compared to 1.8 g/L from monocultures. Pretreatment conditions were optimised using Response Surface Methodology, identifying optimal parameters of 87.5 °C, 3% HCl, and 90 minutes, yielding 4.6 g/L biomass and 2.67 g/L protein. Subsequently, fermentation parameters, including waste-to-water ratio (1:3-1:12), agitation speed (120–180 rpm), co-culture ratio (1:1:1 to 2:1:1), pH (3-7), and retention time (2–8 days) were systematically optimised. Optimal conditions (1:6 waste-to-water ratio, 160 rpm, pH 6, and 7-day fermentation) achieved a maximum biomass of 15.23 ± 1 g/L and protein content of 8.02 ± 0.5 g/L. Scale-up validation through 30-day continuous fermentation demonstrated industrial feasibility, producing a peak biomass of 17 g/L with a protein content of 64% on Day 7. Modified Stover–Kincannon kinetic modelling confirmed efficient substrate utilisation and reactor scalability. These results demonstrate that synergistic acid pretreatment and optimised yeast co-culture systems substantially improve SCP production from agricultural waste. The proposed process provides a sustainable pathway for converting food waste into high-quality protein-rich animal feed, supporting waste valorisation, environmental protection, and global food security.