Background Surgical treatment alone is not effective in addressing delayed fracture healing (DFH). This study nvestigates the molecular mechanism underlying fracture healing to identify improved therapeutic strategies. Methods Serum samples were collected from 76 normal fracture healing (NFH) and 70 DFH patients. RT-qPCR was performed to determine LINC00662, miR-330-3p, and PTEN mRNA expression in serum and Human Bone marrow mesenchymal stem cells (HBMSCs). The diagnostic potential of LINC00662 and miR-330-3p for DFH patients was evaluated via ROC analysis. Binding sites were predicted using bioinformatic databases and validated by dual-luciferase reporter assays. HBMSCs proliferation, apoptosis and osteogenic differentiation were analyzed using CCK-8, flow cytometry, andosteogenesis-related gene expression assays. Results LINC00662 was upregulated and miR-330-3p downregulated in DFH, and the two combined could efficiently diagnose DFH. LINC00662 knockdown promoted runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), osteopontin (OPN), and alkaline phosphatase (ALP) mRNA expression, enhanced HBMSCs proliferation, and suppressed apoptosis. miR-330-3p inhibition reversed these effect. As a target of miR-330-3p, PTEN downregulation ameliorated the negative effects of miR-330-3p downregulation on HBMSCs. Conclusion LINC00662 and miR-330-3p synergistically could improve the diagnostic efficiency of DFH. LINC00662 impedes fracture healing by suppressing osteogenic differentiation and proliferation of HBMSCs while promoting apoptosis in HBMSCs via the miR-330-3p/PTEN axis.