The main objective of this work is to show the proof of concept of a new optofluidic method for high throughput fluorescence-based thermometry, which enables the measure of temperature inside optofluidic microsystems at the millisecond (ms) time scale (high throughput). We used droplet microfluidics to produce highly monodisperse microspheres from dispersed zinc oxide (ZnO) nanocrystals and doped them with rhodamine B (RhB) or/and rhodamine 6G (Rh6G). The fluorescence intensities of these two dyes are known to depend linearly on temperature but in two opposite manner. Their mixture enables for the construction of reference probe whose fluorescence does not depend practically on temperature. The use of zinc oxide microparticles as temperature probes in microfluidic channels has two main advantages: (i) avoid the diffusion and the adsorption of the dyes inside the walls of the microfluidic channels and (ii) enhance dissipation of the heat generated by the focused incident laser beam thanks to the high thermal conductivity of this material. Our results show that the fluorescence intensity of RhB decreases linearly with increasing temperature at a rate of about −2.2%/°C, in a very good agreement with the literature. In contrast, we observed for the first time a nonlinear change of the fluorescence intensity of Rh6G in ZnO microparticles with a minimum intensity at a temperature equal to 40 °C. This behaviour is reproducible and was observed only with ZnO microparticles doped with Rh6G.