Significant clinical challenges encountered in the effective long-term treatment of osteochondral defects have inspired advancements in scaffold-based tissue engineering techniques to aid repair and regeneration. This study reports the development of a biphasic scaffold produced via a rational combination of silk fibroin and bioactive ceramic with stratified properties to satisfy the complex and diverse regenerative requirements of osteochondral tissue. Structural examination showed that the biphasic scaffold contained two phases with different pore morphologies to match the cartilage and bone segments of osteochondral tissue, which were joined at a continuous interface. Mechanical assessment showed that the two phases of the biphasic scaffold imitated the load-bearing behaviour of native osteochondral tissue and matched its compressive properties. In vitro testing showed that different compositions in the two phases of the biphasic scaffold could direct the preferential differentiation of human mesenchymal stem cells towards the chondrogenic or osteogenic lineage. By featuring simple and reproducible fabrication and a well-integrated interface, the biphasic scaffold strategy established in this study circumvented the common problems experienced with integrated scaffold designs and could provide an effective approach for the regeneration of osteochondral tissue.