Abstract Constructing bionic extracellular matrix (ECM) is an attractive proposition for tissue engineering and clinical regeneration therapy involving the stemness of stem cells. Here, a novel recombinant protein fibronectin-collagen peptide (FCP) was designed to modulate the function of ECM expressed by Picha. pastoris strain X33. This FCP promotes cell migration and adhesion and maintains rBMSC stemness by binding integrin β3. Its effects were blocked by both integrin β3 siRNA and the integrin β3 inhibitor Cilengitide. A template-independent ab initio prediction modeling approach is the best approach to construct a stable FCP protein model, which predicts the binding sites between FCP and integrin β3. FCP may be used in the in vitro culture and clinical regeneration of stem cells that highly express integrin β3, such as hematopoietic stem cells. The study provides information on the molecular structure of FCP and its bioactivity, which can be used to design new compounds. Key points • Design a novel recombinant fibronectin-collagen peptide biomimetic ECM. • FCP promotes cell adhesion, migration, and proliferation. • Predicted and verified FCP structure and affinity with integrin β3. • FCP binds integrin β3 to maintain rBMSC stemness.