The culture medium serves as the fundamental basis for recombinant Escherichia coli (E. coli) fermentation, where its composition and formulation critically govern cellular growth, the expression efficiency of target proteins (e.g., collagen), and the cost-effectiveness of industrial-scale production. This study aimed to optimize the culture medium for recombinant E. coli fermentation to improve collagen production efficiency while reducing costs. By conducting single-factor experiments, the key medium components were systematically refined, followed by the application of response surface methodology (RSM) to refine the composition of the culture medium, aiming to enhance production efficiency while reducing costs. The optimized fermentation medium formula obtained was composed of 2.85 g/L glucose, 53.85 g/L complex nitrogen source, and 64.63 mmol/L phosphate buffer. Validation experiments based on this refined formulation demonstrated that the collagen yield reached an impressive 8.62 mg/50 mL, matching that of conventional TB medium while reducing production costs by 30%, highlighting significant economic advantages. A critical innovation was the complete replacement of glucose with straw hydrolysate, which achieved 93% of the original yield, not only confirming the practicality and cost-effectiveness of utilizing agricultural waste as a sustainable feedstock but also providing robust empirical evidence to support the broader adoption of lignocellulosic biomass as a sustainable substitute for starch-derived sugars in microbial fermentation processes. Additionally, scale-up studies conducted at the 5 L bioreactor level unequivocally validated the excellent stability and scalability of the optimized medium, as both bacterial growth kinetics and collagen expression profiles remained consistently unaffected, indicating seamless transition from laboratory to pilot-scale operations. Collectively, these findings establish a solid technical foundation for the high-efficiency, low-cost biological manufacturing of collagen using recombinant E. coli as the host system, offering a transformative pathway toward more sustainable, resource-efficient bioproduction that aligns with circular economy principles while significantly advancing industrial applications in the field of recombinant protein synthesis.