This study introduces a novel retinal temperature determination method based on the temperature dependent properties of photoresponses recorded by electroretinography (ERG). The kinetics and amplitudes of ERG photoresponses depend on retinal temperature. Additionally, raising retinal temperature increases the probability of long-wavelength photon absorption, which manifests as temperature dependence of photoreceptor sensitivity. In this study we extract a number of features that represent these properties from the a- and b-waves of mouse ex vivo ERG flash responses and construct three multivariable regression models between temperature and the selected features. The performance of these models was evaluated against a separate test dataset and for two of the models, an RMS temperature determination error of less than 0.50 °C could be reached. Our results demonstrate that the method can be successfully used for reliable retinal temperature determination ex vivo. The method, reflecting the temperature of distal retina, can be applied also in the estimation of retinal pigment epithelium temperature.