With the increasing applications of 3D printing technology in biomedical field, the composition or additives of the related materials has become critical for the next development. In the current study, we have prepared 3D printed polycaprolactone-hydroxyapatite (PCL-HA) porous scaffolds with loaded heparan sulfate (HS), in order to reveal the reparative effect of different concentrations of HS on the healing of bone defects. As a result, the scaffold itself showed sound compression resistance, air porosity and good biocompatibility. From both in vitro and in vivo experiments, the scaffold with low concentration of HS led to positive effects in promoting osteoblast maturation and accelerating bone defect repair. Moreover, scaffold with high concentration of HS showed notable inhibitive effort on the proliferation of osteoblasts, yet it still brought about positive effects in repairing bone defects in organisms. Thus, with good structural properties and biocompatibility, 3D printed PCL-HA-HS composite scaffold can facilitate and accelerate the repairing of biological bone defects, demonstrating as an effective biomaterial for bone defect repair.