Abstract: The inconsistency of rotational temperature and vibrational temperature in the rarefied flow field is a concrete manifestation of thermodynamic non-equilibrium. The non-intrusive measurement method of rotational temperature and vibrational temperature in the rarefied flow field can be measured by the Electron Beam Fluorescence (EBF) technique. The basic principle and measurement method of EBF were introduced in this paper. Experiment was carried out in the Ma = 12 and Ma = 16 conical nozzle of the wind tunnel. The rotational temperature maximum relative uncertainty is 0.26% and the vibrational temperature maximum relative uncertainty is 0.8% from the analysis of repetitive measurement results. The distribution of rotational temperature and vibrational temperature on the exit section of the Ma = 12 and Ma = 16 conical nozzle reflects the characteristics of expansion flow of the conical nozzle. The measurement results of three states of each nozzle show that with the increase of rareness, the larger the deviation between the vibrational temperature and the rotational temperature is, the more prominent the thermodynamic non-equilibrium phenomenon appears.