Electrospinning is a versatile technology for the fabrication of nanofibrous matrixes to regenerate defects. This study aims to develop a functionalized and electroconductive polymeric matrix to improve rat bone marrow mesenchymal stem cell adhesion, proliferation, and differentiation. Herein, the influence of the chemical composition of the substrate on homogeneous modification of the surface with mussel-inspired polydopamine (PDA) is focused. Accordingly, the deposition of PDA on the surface was proved by Fourier transform infrared spectroscopy. Morphologies of the scaffolds demonstrated homogeneous decoration of the polyvinyl alcohol (PVA)/polyurethane (PU)-polyaniline (PANI) matrixes with PDA, while a lower density of mussel-inspired polymer was observed in bare PU-PANI constructs. Although uniform and dense precipitation of PDA reduced conductivity of scaffolds 1.2 times compared with the samples with a low density of the PDA, 1.1 and 1.2 times enhancement in tensile strength and Young's modulus, respectively, were the strength of the applied process, especially in bone tissue engineering area. Contact angle measurements demonstrated about two times reduction in measured values, which shows improvement in hydrophilicity of PDA-modified PVA/PU-PANI fibers compared with PDA-coated PU-PANI ones. Swelling ratio and mass loss ratio calculations revealed enhancement in measured values as a function of homogeneous and dense coating, which arise from hydrophilicity of the polymeric substrate. The bioactivity test indicated that a dense layer of PDA strongly supports formations of hydroxyapatite-like crystals. Moreover, homogeneous decoration of conductive matrixes with PDA showed suitable cell viability, adhesion, and spreading while cellscaffolds interactions improved under electrical stimulation. Higher expression of alkaline phosphatase and secretion of Collagen I under the electrical field proved the applicability of modified electroconductive scaffolds for further preclinical and clinical studies to introduce as a reconstructive bone substitute.