Recombinant human collagens have attracted intensive interest in the past two decades, demonstrating considerable potential in medicine, tissue engineering, and cosmetics. Several humanized recombinant collagens have been produced, exhibiting similar characteristics as the native species. To get insight into the structural and bioactive properties of different parts of collagen, in this study, the segment of Gly300-Asp329 of type III collagen was first adopted and repeated 18 times to prepare a novel recombinant collagen (named rhCLA). RhCLA was successfully expressed in E. coli, and a convenient separation procedure was established through reasonably combining alkaline precipitation and acid precipitation, yielding crude rhCLA with a purity exceeding 90%. Additionally, a polishing purification step utilizing cation exchange chromatography was developed, achieving rhCLA purity surpassing 98% and an overall recovery of approximately 120 mg/L culture. Simultaneously, the contents of endotoxin, nucleic acids, and host proteins were reduced to extremely low levels. This fragmented type III collagen displayed a triple-helical structure and gel-forming capability at low temperatures. Distinct fibrous morphology was also observed through TEM analysis. In cell experiments, rhCLA exhibited excellent biocompatibility and cell adhesion properties. These results provide valuable insights for functional studies of type III collagen and a reference approach for the large-scale production of recombinant collagens.