Application of traditional electrospun scaffolds in tissue engineering is limited due to the sheet-like nanofiber layers hindered cell infiltration. In this study, a three-dimensional (3D) thick nanofiber stack with pore size gradient was fabricated via a novel electrospinning setup. Instead of a traditional flat-plate collector, a small copper plate which covered a hole of PMMA sheet was connected to negative voltage, resulting in focused collection of nanofibers and quickly enhanced scaffold thickness due to insulating PMMA preventing fibers from spreading elsewhere. The average pore size can reach more than 25 mu m which benefit for cell infiltration. In the process of electrospinning, the mat with increased thickness may influence the electric field and has effect on fiber diameter and pore size. (C) 2017 Published by Elsevier B.V.