Injury to articular cartilage, especially the defects induced by degenerative diseases has presented insurmountable challenges. Elaborating a replacement of articular cartilage using biomimic tissue-engineering strategies provides a promising remedy. However, none of the previous osteo/chondrogenic methodologies can not only simultaneously induce osteo/chondrogenesis of stem cells in one scaffolding niche, but also generate a biomimic interface between the formed osteogenic and chondrogenic zones. We report here an innovative method using biomicrofluidic techniques to simultaneously steer distinct specialized differentiation of stem cells into chondrocytes and osteoblasts in one hydrogel slab. Importantly, a gradient that mimics the interface of bone-to-cartilage was generated in the middle of the hydrogel slab. We compared this format with the conventional method for osteochondrogenesis; this format using the gradient-generating microfluidic device indicated outstanding superiorities in stem cell culture and differentiation. Our findings will have a major impact on the design of versatile biomicrofluidic devices for interfacial tissue regeneration.