Objective: To construct a two-dimensional composite membrane and a three-dimensional biomimetic scaffold by silk fibroin (SF), type I collagen (Col-I) and hydroxyapatite (HA) blends in vitro, and to study its physical and chemical properties and biocompatibility. To explore the feasibility of its application in tissue engineering scaffold materials. Methods: Two-dimensional composite membranes and three-dimensional scaffolds were prepared by blending SF/COL-I/HA at the bottom of the cell culture chamber and low temperature 3D printing combined with vacuum freeze drying. The biocompatibility was evaluated by mechanical properties testing, electron microscopy and Micro-CT to examine the physicochemical properties of the material and to detect cell proliferation. Results: Stable two-dimensional composite membrane and three-dimensional porous structural scaffolds were obtained by blending and low temperature 3D printing. The mechanical properties were consistent. The pore size, water absorption, porosity and elastic modulus were all in accordance with the requirements of constructing tissue engineering bone. The scaffold is a grid-like white cube with good internal pore connectivity; hydroxyapatite (HA) is evenly distributed in the composite membrane, and the cells are attached to the composite membrane in a flat shape; the cells are distributed around the pore walls of the scaffold. The shape of the shuttle is good, and the growth and proliferation are good. Conclusion: In this study, the composite membrane and three-dimensional scaffold prepared by SF/col-I/HA blending system have better pore connectivity and pore structure, which is beneficial to cell and tissue growth and nutrient transport. Its physical and chemical properties and biocompatibility meet the requirements of bone tissue engineering biomaterials. Related research provides important experimental data and theoretical basis for the application and evaluation of bone tissue engineering scaffold materials, which is of great significance for clinically realizing the repair and regeneration of human tissue and organ defects.