Endogenous electric fields (EFs) play an important role in many biological processes. In order to gain an insight into these biological phenomena, externally applied electric fields are used to study cellular responses. In this work, we report the construction and fabrication of a direct current (DC)-electrically stimulated microfluidic biochip and its validation with murine photoreceptor-derived 661 W cells. The presented device has the particularity of offering a non-homogeneous EF environment that best resembles the endogenous electric fields in vitro. The fabrication process is relatively easy, namely by photolithography and soft lithography techniques and, furthermore, it enables live-cell imaging under an inverted microscope. First experimental results reveal cathodal directional cell migration upon applied DC EFs. In summary, the microfluidic biochip has proven biocompatibility and suitability for cellular electrotaxis experiments in non-homogeneous DC electric fields.