Electroosmotic flows (EOFs) on insulated interfacial surface commonly exists as interfacial flows. Previously theoretical studies indicate that EOFs of Newtonian fluids on the insulated interfacial surface are steady in microchannels with symmetric zeta potentials (Suresh and Homsy, Physics of Fluids, 2004, 16, 2,349). Restricted by flow diagnostic methods in microfluidics, few velocity measurements of instantaneous EOFs have been reported, and the existence of unsteady EOFs on the insulated surface remains unclear. In this investigation, the velocity fluctuations of EOFs generated under AC electric field (named as ACFEOF) overlapped on a steady pressuredriven flow are measured by laser induced fluorescence photobleaching anemometer, at the diffuse electric double layer (EDL) on the bottom wall far from electrodes. Chaotic velocity fluctuations according to unsteady ACFEOF has been, for the first time, observed. Stokes number (St) and electrical Reynolds number (Re E) related to oscillation and electro-inertial effect are suggested to control chaotic ACFEOF.