A polarization fluorometer and a method for calibrating such a fluorometer. The fluorometer includes an excitation channel with an excitation polarizer for generating light polarized in a first plane or in a second plane to be conducted as a excitation light to a sample, an emission channel for conducting emission light from the sample, in which emission channel there is an emission polarization filter, an excitation detector, to which light polarized by the excitation polarizer can be conducted from the excitation channel, and a reference detector, to which light polarized by the excitation polarizer can be conducted from the excitation channel before being conducted to the sample or to the excitation detector. The reference detector can be calibrated by the excitation detector so that correction measurements can be carried out in real time during sample measurement. The fluorometer can be used especially for fluorescence spectra measurements.