Alternating current electrothermal flow (ACET) induced by Joule heating is utilized to transport biologically relevant liquids in microchannels using simple electrode designs. However, Joule heating may cause significant temperature rises, which can degrade biological species, and hence, ACET may become impractical for biomicrofluidic sensors and other possible applications. In this study, the temperature rise at the electrode/electrolyte interface during ACET flow is measured using a high-resolution, noninvasive, thermoreflectance imaging method, which is generally utilized in microelectronics thermal imaging applications. The experimental findings reveal that Joule heating could result in an excessive temperature rise, exceeding 50 °C at higher voltage levels (20 Vpp). The measured data are compared with the results of the enhanced ACET theoretical model, which predicts the temperature rise accurately, even at high levels of applied voltages. Overall, our study provides a temperature measurement technique that is used for the first time for electrode/electrolyte systems. The reported results are critical in designing biomicrofluidic systems with significant energy dissipation in conductive fluids.