With the present trend toward a miniaturization of chemical systems comes the need for a precise characterization of physicochemical parameters in very small fluid volumes. We describe here an original approach for small-scale temperature measurements based on the detection of fluorescent protein blinking. We observed that the characteristic time associated with the reversible protonation reaction responsible for the blinking of the enhanced green fluorescent protein is strongly temperature dependent at low pH. The blinking characteristic time can easily be detected by fluorescence correlation spectroscopy, and therefore provides the means for noninvasive, spatially resolved, absolute temperature measurements. We applied this approach to the quantification of laser-heating effects in thin liquid samples. As expected, we observed a linear dependence between the temperature increase at the laser focus and both the laser power and the sample extinction coefficient. In addition, we were able to measure the la...