In recent years, three-dimensional (3D) scaffolds have proven to be highly advantageous in mammalian cell culture and tissue engineering compared to 2D substrates. Herein, we demonstrated the fabrication of novel 3D core-shell nanofibers (3D-CSNFs) using an improved electrospinning process combined with in situ surface polymerization. The obtained 3D nanofibrous scaffold displayed excellent mechanical and electrical properties. Moreover, the cotton-like 3D structure with large internal connected pores (20-100 mu m) enabled cells to easily infiltrate into the interior of the 3D scaffold with a good spatial distribution to mimic the ECM-like cell microenvironments. Stable cell-fiber composite constructs were formed in the 3D-CSNFs with relatively higher adhesion and viability compared to 2D-CSNFs. Furthermore, the human mesenchymal stem cells (hMSCs) cultured on conductive polymer coated electrically active 3D nanofibers responded with a healthy morphology and anchorage on the fibers with relatively higher viability and proliferation under electrical stimulation (ES). This study demonstrates the successful fabrication of 3D-CSNFs and the constructive interaction of the 3D microenvironment and subsequent electrical stimulations on hMSCs, thereby holding promising potential in tissue engineering and regenerative therapies aided by electro-stimulation-based differentiation strategies.