Abnormal heart valve function is a major cause of heart disease and is often associated with high rates of morbidity and mortality from heart disease. Focusing on the field of valve replacement, tissue-engineered heart valves are becoming popular and attracting more attention. In this work, an artificial heart valve scaffold based on polycaprolactone/polyurethane (PCL/PU) three-layer composite fibers was prepared by 3D printing and electrospinning, including an inner PCL layer, a middle PCL/PU layer, and an outer PCL layer. The obtained valve scaffold had strong circumferential mechanical properties when PCL/PU = 1:2, and its elastic modulus was 14.7 MPa, similar to that of natural valve. The heart valve scaffold had good cytocompatibility, and the final cell survival rate was 99.8% after 14 days of cell culture. The layered structure makes the artificial heart valve more similar to the natural structure of the mammalian heart, which is conducive to cell proliferation and differentiation, and provides a reference solution for interventional treatment of heart valves.