Objective To construct a two-dimensional (2D) composite membrane and a three-dimensional (3D) biomimetic scaffold by silk fibroin (SF), type I collagen (Col-I) and hydroxyapatite (HA) blends in vitro, so as to study its physicochemical properties, as well as biocompatibility and explore the feasibility of its application in tissue engineering scaffold materials. Methods 2D composite membranes and 3D scaffolds were prepared by blending SF/Col-I/HA at the bottom of cell culture chamber and low temperature 3D printing combined with vacuum freeze drying. The biocompatibility was evaluated by mechanical property testing, scanning electron microscope and Micro-CT to examine the physicochemical properties of the material, and cell proliferation was detected to evaluate its biocompatibility. Results Stable 2D composite membrane and 3D 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 was a grid-like white cube with good internal pore connectivity; HA was evenly distributed in the composite membrane, and the cells were attached to the composite membrane in a flat shape; the cells were distributed around pore walls of the scaffold. The shape of the shuttle was fusiform, and the growth and proliferation were good. Conclusions The composite membrane and 3D scaffold prepared by SF/Col-I/HA blending system had better pore connectivity and pore structure, which was beneficial to cell and tissue growth and nutrient transport. Its physicochemical properties and biocompatibility could meet the requirements of bone tissue engineering biomaterials.