The temperature dependent quantum efficiency of fluorescent dyes can be used to measure changes in temperature of the fluorescent dye molecules. This paper describes the use of Rhodamine dye as a temperature sensitive based fluorescence indicator for the characterization of heating in a pressure driven microfluidic device with high spatial resolution. A microheating element is used to control the temperature of the fluid in order to create localized heating in the microchannel. The temperature dependent fluorescence intensity of Rhodamine 6G for a concentration of 1 mmol / L is characterized with a 5th order polynomial then used to detect the temperature of the fluid in a microchannel. Fluid temperatures in the range of 20-85 ° C are measured with an uncertainty of < 2 ° C using a confocal fluorescence microscope. This method enables the characterization, imaging and mapping of three-dimensional temperature gradients within a microchannel.