In this paper we present a numerical and experimental investigation of a chaotic mixer in a microchannel via low frequency switching transverse electroosmotic flow. By applying a low frequency, square-wave electric field to a pair of parallel electrodes placed at the bottom of the channel, a complex 3D spatial and time-dependence flow was generated to stretch and fold the fluid. This significantly enhanced the mixing effect. The mixing mechanism was first investigated by numerical and experimental analysis. The effects of operational parameters such as flow rate, frequency, and amplitude of the applied voltage have also been investigated. It is found that the best mixing performance is achieved when the frequency is around 1 Hz, and the required mixing length is about 1.5 mm for the case of applied electric potential 5 V peak-to-peak and flow rate 75 µL h−1. The mixing performance was significantly enhanced when the applied electric potential increased or the flow rate of fluids decreased.