A technique for measuring surface temperature field in micro scale is newly proposed, which uses temperature-sensitive fluorescent (TSF) dye coated on the surface and is easily implemented with a fluorescence microscope and a CCD camera. The TSF dye is chosen among mixtures of various chemical compositions including rhodamine B as the fluorescent dye to be most sensitive to temperature change. In order to examine the effectiveness of this temperature measurement technique, numerical analysis and experiment on transient conduction heat transfer for two different substrate materials, i. e., silicon and glass, are performed. In the experiment, to accurately measure the temperature with high resolution temperature calibration curves were obtained with very fine spatial units. The experimental results agree qualitatively well with the numerical data in the silicon and glass substrate cases so that the present temperature measurement method proves to be quite reliable. In addition, it is noteworthy that the glass substrate is more appropriate to be used as thermally-insulating locally-heating heater in micro thermal devices. This fact is identified in the temperature measuring experiment on the locally-heating heaters made on the wafer of silicon and glass substrates. Accordingly, this technique is capable of accurate and non-intrusive high-resolution measurement of temperature field in microscale.