In this study, recombinant human bone morphogenetic protein-2 (rhBMP-2) delivery system with slow mode was successfully developed in three-dimensional (3D) printing-based polycaprolactone (PCL)/poly(lactic-co-glycolic acid) (PLGA) scaffolds for bone formation of critical-sized rabbit segmental diaphyseal defect. To control the delivery of the rhBMP-2, collagen (for long-term delivery up to 28 days) and gelatin (for shor-term delivery within a week) solutions encapsulating rhBMP-2 were dispensed into a hollow cylinderical type of PCL/PLGA scaffold. An effective dose of 5μg/mL was determined by measuring the alkaline phosphatase and osteocalcin gene expression levels of human nasal inferior turbinate-derived mesenchymal stromal cells (hTMSCs) seeded on the PCL/PLGA/collagen scaffold in vitro. However, it was found that a burst release of rhBMP-2 from the PCL/PLGA/gelatin scaffold did not induce the osteogenic differentiation of hTMSCs in vitro at an equivalent dose. In the in vivo animal experiements, microcomputed tomography and histological analyses confirmed that PCL/PLGA/collagen/rhBMP-2 scaffolds (long-term delivery mode) showed the best bone healing quality at both weeks 4 and 8 after implantation without inflammatory response. On the other hand, a large number of macrophages indicating severe inflammation provoked by burst release of rhBMP-2 were observed in the vicinity of PCL/PLGA/gelatin/rhBMP-2 (short-term delivery mode) at week 4.