Electro-osmotic flow (EOF) instability in a microchannel has been experimentally investigated using a micro-PIV system. The micro-PIV system consisting of a two-head Nd:Yag laser and cooled CCD camera was used to measure instantaneous velocity fields and vorticity contours of the EOF instability in a T-shape glass microchannel. The electrokinetic flow instability occurs in the presence of electric conductivity gradients. Charge accumulation at the interface of conductivity gradients leads to electric body forces, driving the coupled flow and electric field into an unstable dynamics. The threshold electric field above which the flow becomes unstable and rapid mixing occurs is about 1000V/cm. As the electric field increases, the flow pattern becomes unstable and vortical motion is enhanced. This kind of instability is a key factor limiting the robust performance of complex electrokinetic bio-analytical devices, but can also be used for rapid mixing and effective flow control fer micro-scale bio-chips.