The invention relates to the field of field emission materials, and aims at providing the preparation of nano single crystal lanthanum hexaboride and the application of the nano single crystal lanthanum hexaboride in the electron microscope filament preparation. The preparation comprises the following steps: weighing lanthanum compounds and alkali metal boron hydride powder, and performing ball-milling to obtain a mechanical mixture; placing the mechanical mixture into a reactor, heating up to 300 to 700 DEG C from the room temperature at the heating rate of 2 DEG C per minute, then performing heat preservation for 2 hours and releasing hydrogen; then cooling, washing, separating and performing vacuum drying at the temperature of 80 DEG C to obtain the nano single crystal lanthanum hexaboride. According to the preparation, the synthesis temperature of lanthanum hexaboride is greatly decreased, the quality management during the industrial production of the lanthanum hexaboride is facilitated, the single crystal particle size consistency of the lanthanum hexaboride is improved, and the quality management of large-scale production is facilitated. The raw material cost is low, the energy consumption is low, and the preparation process is easy and simple. When the nano single crystal lanthanum hexaboride is used for preparing an electron microscope filament, low work function, low volatility and low resistance are realized; the mechanical strength is high, and the chemical stability is good, so that an electron microscope image is more clear, the cost is lower and the service life is prolonged greatly.