We demonstrate an artificial three-dimensional (3D) electrical active human neuronal network system, by the growth of brain neural progenitors in highly porous low density electrospun poly-ε-caprolactone (PCL) fiber scaffolds. In neuroscience research cell-based assays are important experimental instruments for studying neuronal function in health and disease. Traditional cell culture at 2D-surfaces induces abnormal cell–cell contacts and network formation. Hence, there is a tremendous need to explore in vivo-resembling 3D neural cell culture approaches. We present an improved electrospinning method for fabrication of scaffolds that promote neuronal differentiation into highly 3D integrated networks, formation of inhibitory and excitatory synapses and extensive neurite growth. Notably, in 3D scaffolds in vivo-resembling intermixed neuronal and glial cell network were formed, whereas in parallel 2D cultures a neuronal cell layer grew separated from an underlying glial cell layer. Hence, the use of the 3D cell assay presented will most likely provide more physiological relevant results.