Three-dimensional elastic nanofibers (3D eNFs) can offer a suitable 3D dynamic microenvironment and sufficient flexibility to regulate cellular behavior and functional protein expression. In this study, we report a novel approach to prepare 3D nanofibers with excellent mechanical properties by solution-assisted electrospinning technology and in situ polymerization. The obtained 3D eNFs demonstrated excellent biocompatible properties to meet cell culture requirements under a dynamic environment in vitro. Moreover, these 3D eNFs also promoted human bone marrow mesenchymal stem cells (hMSCs) adhesion and collagen expression under biomechanical stimulation. The results demonstrated that this dynamic cell culture system could positively impact cellular collagen but has no significant effect on the proliferation of hMSCs grown in the 3D eNFs. This work may give rise to a new approach for constructing a 3D cell culture for tissue engineering.