Background: Tricalcium phosphate (TCP, Molecular formula: Ca3(PO4)2) is a hydrophilic bone graft biomaterial extensively used for guided bone regeneration (GBR). However, few studies have investigated 3D-printed polylactic acid (PLA) combined with the osteo-inductive molecule fibronectin (FN) for enhanced osteoblast performance in vitro, and specialized bone defect treatments. Aim: This study evaluated PLA properties and efficacy following glow discharge plasma (GDP) treatment and FN sputtering for fused deposition modeling (FDM) 3D printed PLA alloplastic bone grafts. Methods: 3D trabecular bone scaffolds (8 × 1 mm) were printed by the 3D printer (XYZ printing, Inc. 3D printer da Vinci Jr. 1.0 3-in-1). After printing PLA scaffolds, additional groups for FN grafting were continually prepared with GDP treatment. Material characterization and biocompatibility evaluations were investigated at 1, 3 and 5 days. Results: SEM images showed the human bone mimicking patterns, and EDS illustrated the increased C and O after fibronectin grafting, XPS and FTIR results together confirmed the presence of FN within PLA material. Degradation increased after 150 days due to FN presence. 3D immunofluorescence at 24 h demonstrated better cell spreading, and MTT assay results showed the highest proliferation with PLA and FN (p< 0.001). Cells cultured on the materials exhibited similar alkaline phosphatase (ALP) production. Relative quantitative polymerase chain reaction (qPCR) at 1 and 5 days revealed a mixed osteoblast gene expression pattern. Conclusion: In vitro observations over a period of five days, it was clear that PLA/FN 3D-printed alloplastic bone graft was more favorable for osteogenesis than PLA alone, thereby demonstrating great potential for applications in customized bone regeneration.