Articular cartilage is refractory to self-healing due to the absence of vascular, nervous, and lymphatic systems, and its repair remains a clinical challenge. Tissue regeneration through recruitment of stem cells cell-free scaffolds is a promising alternative strategy. Herein, a kind of functional injectable hydrogel system (Col-Apt@KGN MPs), which is a collagen-based and microsphere-embedded cell-free scaffold, was designed to achieve spatiotemporal regulation of endogenous mesenchymal stem cells (MSCs) recruitment and their chondrogenic differentiation by respective release of aptamer 19S (Apt19S) and kartogenin (KGN). results confirmed that the Col-Apt@KGN MPs hydrogel had sequential release characteristics. Apt19S was rapidly released from the hydrogel within 6 days, while KGN was slowly released for 33 days the degradation of poly(lactic--glycolic acid) (PLGA) microspheres. When cultured with MSCs, the Col-Apt@KGN MPs hydrogel supported the adhesion, proliferation, and chondrogenic differentiation of MSCs. results indicated that the Col-Apt@KGN MPs hydrogel effectively promoted the recruitment of endogenous MSCs in a rabbit full-thickness cartilage defect model; furthermore, the Col-Apt@KGN MPs hydrogel enhanced the secretion of cartilage specific extracellular matrix and achieved the reconstruction of subchondral bone. This study demonstrates that the Col-Apt@KGN MPs hydrogel possesses great potential in recruitment of endogenous stem cells and cartilage tissue regeneration.