Microscale temperature mapping in liquids is of great importance in many areas of research such as microfluidics and biology. Among the current thermometric approaches, optical probing using fluorescence is particularly desirable because of its high spatial resolution and non-invasive nature. Here we report a new microscale two-dimensional (2D) fluorescence thermometry. This method exploits the temperature dependence of rotational molecular motion and its influence on the depolarization of fluorescence light, by measuring the difference in fluorescence intensities excited by orthogonal polarizations. With one charge coupled device (CCD) camera, we get 2D ratiometric mappings of temperature from successively recorded fluorescence images under alternatively polarized excitations. We demonstrate reliable temperature mapping in liquids at sub-1 °C temperature accuracy and sub-10 µm spatial resolution. We also show that the proposed thermometry approach is robust against fluorescence intensity variations, suitable for 2D mapping and of fast readout that is comparable to CCD framerate. Moreover, it is easy to be integrated into microscope systems since only rotation of excitation polarization is needed.