Background: Drug resistance of pathogens and immunosuppression are the main causes of clinical stagnation of osteomyelitis. The ideal treatment strategy for osteomyelitis is to achieve both efficient antibacterial and bone healing through spatiotemporal modulation of immune microenvironment. Methods: In this study, a bilayer hydrogel based on genetically engineered polypeptide AC(10)A and AC(10)A RGD was prepared by self-assembly. Ag2S QDs@DSPE-mPEG(2000)-Ce6/Aptamer (AD-Ce6/Apt) was loaded in the top layer AC(10)A hydrogel (AA) for antibacterial, and bone marrow-derived mesenchymal stem cells (BMSCs) were loaded in the lower layer AC(10)ARGD hydrogel (MAR) for bone healing. The AD-Ce6/Apt can be released from the AA hydrogel to target S. aureus before bacterial biofilm formation and achieved significant bactericidal effect under irradiation with a 660 nm laser. Moreover, AD-Ce6/Apt can induce M1 type polarization of macrophages to activate the immune system and eliminate residual bacteria. Subsequently, BMSCs released from the MAR hydrogel can differentiate into osteoblasts and promote the formation of an anti-inflammatory microenvironment by regulating the M2 type polarization of macrophages. The bilayer AA-MAR hydrogel possessed good biocompatibility. Results: The in vitro and in vivo results showed that the AA-MAR hydrogel not only realized efficient photodynamic therapy of S. aureus infection, but also promoted the transformation of immune microenvironment to fulfill the different needs of each stage, which ultimately improved bone regeneration and mechanical properties post-surgery. Conclusion: This work presents an approach for spatiotemporal modulation of immune microenvironment in the treatment of osteomyelitis. [GRAPHICS] .