In ex vivo gene therapy, tissue engineering scaffolds have been widely applied as gene carriers. As a class of scaffolds with different surface topographies for tissue engineering, scaffolds with aligned fibre architecture had been proven to enhance cell proliferation and migration. However, the influence of scaffolds' fibre alignment on gene therapy is still unknown. This study investigated the osteogenic potential of different fibre arrangement scaffolds seeded with BMP2-transfected human periodontal ligament stem cells (BMP2/PDLSCs), aiming to explore whether gene therapeutic effects could be influenced by aligned scaffolds. BMP2/PDLSCs were cultured on scaffolds with different surface topographies: two kinds of aligned scaffolds (parallel and cross scaffolds) and two kinds of unaligned scaffolds as controls (random and smooth films). Cell morphology, proliferation, osteogenesis-related markers were analysed. BMP2/PDLSCs on aligned scaffolds showed elongation along the axes of scaffolds. DNA content, osteogenesis-related gene and protein expression, ALP activity, collagen I and calcium content of BMP2/PDLSCs seeded on aligned scaffolds were significantly higher than that on unaligned scaffolds. Aligned scaffolds influenced the orientation, enhanced the proliferation, and promoted the osteogenic differentiation of BMP2/PDLSCs. Results indicated aligned scaffolds could be used to enhance the biologic effects of ex vivo gene therapy for bone regeneration.