Large complex bone defects pose a significant clinical challenge. Conventional bone grafting approaches cannot simultaneously achieve tissue regeneration and infection prevention, resulting in impaired healing outcomes and prolonged treatment cycles. Existing therapeutic strategies lack integrated solutions capable of concurrently providing infection prevention and osteogenesis promotion within a single platform. This study developed a novel multifunctional composite scaffold using dual-nozzle 3D printing technology to simultaneously achieve infection prevention and accelerated bone regeneration. Linezolid-loaded poly(lactic-co-glycolic acid) microspheres (LMS) were uniformly dispersed within the pores of Calcium sulfate/polylactic acid (CS/PLA) scaffolds to successfully construct the composite scaffold. In vitro characterization revealed uniform distribution of microspheres within the scaffold pores, with the fabricated CS/PLA-LMS demonstrating excellent biocompatibility and mechanical properties, achieving an elastic modulus of 87 MPa. Furthermore, the composite scaffold effectively inhibited Staphylococcus aureus activity in vitro. In vivo studies in a rat cranial defect model revealed that the composite scaffold significantly enhanced bone formation compared to blank controls, bone volume fraction increased by 3.2 times, and trabecular spacing decreased by 50%, with mechanistic analysis indicating activation of the PI3K-AKT signaling pathway. The integrated design successfully prevented infection-related complications while promoting robust osteogenesis, offering a clinically relevant solution for treating complex bone defects where infection prevention and regenerative capacity are primary therapeutic concerns.