The nanofiber surface modified with physical or chemical gradients is very useful in a wide range of areas including tissue engineering, regenerative medicine, drug screening, and biomaterial chemistry. In this work, we presented a novel and straightforward microfluidic assisted approach to produce electrospinning nanofibers containing gradients in different compositions, nanoparticles and biomolecule concentrations. The series of gradient nanofibers were mainly produced by using a two inlet microfluidic device in combination with an electrospinning nozzle on a 3-D controllable platform, which exhibited different functions and properties. The controlled nanofibers with incorporated biomolecule gradient were used for guiding the spatial differentiation in mesenchymal stem cells (MSCs). This established approach is very simple, and flexible to operate, which might find enormous potential for biology and tissue engineering applications.