The invention relates to a method for calibrating substrate surface actual temperature in molecular beam epitaxy. The method comprises the steps of (1), testing a thermocouple temperature T1 in substrate analysis; (2) performing temperature reduction after substrate analysis, after surface reconstruction change is observed and confirmed, stopping a beam source for protecting the substrate, performing temperature increase, observing and recording a thermocouple temperature T2 when the substrate surface reconstruction is restored; (3), reducing the temperature of the substrate to the room temperature, covering a noncrystal layer on the substrate surface, performing temperature increase, observing and recording a thermocouple temperature T3 in desorption of the noncrystal layer; and (4), according to the surface real temperature values of the substrate when the thermocouple temperatures are respectively T1, T2 and T3, establishing a relationship between the substrate surface real temperature and the thermocouple temperature by means of a B spline function. The method can be used for accurately acquiring the actual temperature of the substrate surface in molecular beam epitaxy and has advantages of high speed, simple operation and high accuracy. The method has high application value in researching molecular beam epitaxy material growth dynamics and accurately controlling growth of micro-nano structure material.