Abstract: Fluorescence quantum efficiency is the ratio of the number of photons emitted by fluorescent materials to the number of photons absorbed, which is used to evaluate the luminescence performance of fluorescent materials. In this paper, aiming at the problems of fluorescence quantum efficiency measurement by the absolute method in reference materials preparation, we studied the four-pass cuvette wide-band matching consistency test method, which eliminates the influence of inconsistent transmission ratio of cuvette incident surface on the measurement of fluorescence quantum efficiency. The measurement method of segmented spectrum acquisition was studied to eliminate the influence of excitation on the emission band and ensure the accuracy of emission band integration. The effect of solvent evaporation in the cuvette on fluorescence quantum efficiency was studied. The sealing of the cuvette can eliminate the influence of volume and concentration fluctuations in the measurement of fluorescence quantum efficiency. These studies provide guidance for the accurate measurement of fluorescence quantum efficiency by the absolute method.