This study presents a one-station 3D fabrication technique of nanofibrous scaffold for tissue engineering. A divergence static electric field was introduced in an electrospinning system to induce a self-assembly of aligned polycaprolactone (PCL) nanofibers into a tunable 3D architecture with thickness ranging from 2 to 12 mm. Silver nanoparticles were incorporated into the PCL solution to alter the electrical conductivity. Human fibroblast cells were cultured on the pure PCL nanofiber scaffolds in vitro for 7 days. It was found that the occurrence of nanofiber bridging phenomenon depended on the solution viscosity. The minimum viscosity to form a 3D nanofiber structure was higher than that to form a 2D nanofiber mat. The homogeneity of nanofiber distribution within the 3D space was positively correlated with the electrical conductivity and the weight of the nanofibers. In the cell culture test, fibroblasts proliferated on the scaffold and organized as an aligned matrix which mimicked the microstructure of native musculoskeletal tissues.