The present invention provides a preparation method of a transmission electron microscopy film sample for in-situ application of the electric field and the stress. The preparation method comprises: (1) cutting a film to be detected and a single crystal Si substrate into a strip shape with a size of 3 mm*1 mm*0.5 mm, and adhering the film surface of the film strip and the polishing surface of the Si strip; (2) carrying out mechanical grinding and polishing on both surfaces having the film strip thickness; (3) using a nail thinning instrument to carry out nail thinning; (4) adhering a half copper ring; and (5) selecting an ion thinning instrument to carry out ion thinning, setting the ion gun voltage to 4 kV, setting the angle to 6 DEG, thinning for 20-40 min, changing the angle into 4 DEG, thinning for 15-30 min, and finally polishing for 3 min by adopting the angle of 3 DEG and the voltage of 3 kV so as to obtain the large area perforation on the adhering seam, wherein the large area adhered Si sheet is bombarded, the film is exposed, the electric field and the stress can be applied by using the probe, and the large area thin region suitable for observation by the transmission electron microscopy exists on the edge.