Elastin-like polypeptides (ELPs), a temperature-responsive biological macromolecule, is used to develop a novel and eco-friendly strategy for biotitania synthesis. Three ELPs (V9F, KV8F, and K5V4F) are designed and overexpressed in E. coli. Then these recombinant ELPs are purified with high yields by using phase transition-based green ITC method. ELPs present the capabilities of ultrafast formation of biotitania within seconds. The specific activity of ELPs is significantly influenced by the number of lysine residues. K5V4F is the one with the highest specific activity of 56.90, which is 26.34 times higher than that of R5 peptide. Moreover, ELPs-mediated biomimetic titania mineralization can perform in a broad pH range (2.2 ≤ pH ≤ 10.8) at ambient temperatures, however, the optimum condition is near-neutral pH. The biotitania nanoparticles are evidenced to be as solid, spherical, and amorphous by SEM, TEM, and SAED analysis. Based on the unique temperature-responsiveness of ELPs and their excellent biomimetic mineralization abilities, this method may have great potentials to prepare smart biotitania nanomaterials for various application fields, such as biomedical engineering, biomanufacturing, and environmental remediation.