Abstract: The recombinant elastin-like/squid ring tooth protein SRT-ELP36 integrates the sequence advantages of elastin-like polypeptides and squid ring tooth proteins, exhibiting superior breaking strength and toughness compared to spider silk proteins, and holds great application potential in the field of biomaterials. In this study, Escherichia coli BL21(DE3) was used as the expression host to improve the production of SRT-ELP36 through the optimization of high-density fermentation process. Firstly, single-factor experiments were conducted to screen the nutritional components affecting the expression of SRT-ELP36, and then Plackett-Burman (PB) experiment was used to identify yeast extract, peptone and corn steep liquor powder as the key factors affecting fermentation yield. The weight of each factor was clarified by principal component analysis: the key factors were yeast extract, tryptone and corn steep liquor powder, the secondary key factors were betaine and proline, and the general factors were ammonium sulfate, magnesium sulfate, glycine and potassium chloride. Furthermore, the central composite design (CCD) was utilized to determine the optimal fed-batch medium formulation: 6.8 g/L yeast extract, 7.5 g/L tryptone and 8.0 g/L corn steep liquor powder. Finally, the optimized process was verified in a 50 L bioreactor with the core regulation strategy of optimized fed-batch medium combined with precise control of dissolved oxygen and residual sugar. The results showed that the high-density culture of E. coli was achieved with OD₆₀₀ reaching 100.2, and the volumetric production of SRT-ELP36 reached 2.52 g/L, which was a 25.0% increase compared with that before optimization. SRT-ELP36 existed as inclusion bodies in the bacterial precipitate, and a standardized SDS-PAGE semi-quantitative detection method was established. This study clarified the key nutritional regulation factors and their weight relationship for the high-density fermentation of SRT-ELP36, and established a scalable fed-batch fermentation process, which laid a technical foundation for the industrial production of SRT-ELP36 recombinant protein.