The invention discloses a preparation method of a holographic observation electron microscope sample. The method comprises the following steps: firstly sputtering a metal weak adsorption layer comprising metal particles with the particle sizes being 10 to 50nm, with the thickness being 100 to 500nm, on the surface of a sample to be tested, or coating an organic glue weak adsorption layer with the thickness being 100 to 500nm, depositing a protective layer containing organics, with the thickness being 500nm to 4 micrometers, on the surface of the metal weak adsorption layer or the organic glue weak adsorption layer, cutting the sample to be tested in a viewing area into transmission slices with the thicknesses being 30 to 150nm through a focusing ion beam, cutting the two ends of the transmission slices to a position of the sample to be tested, so as to obtain a transmission electron microscope sample, finally cleaning an amorphous layer on the surface of the transmission electron microscope sample through the focusing ion beam, and peeling the metal weak adsorption layer or the organic glue weak adsorption layer at the top end of the transmission electron microscope sample and the protective layer containing the organics, so as to form a target product. The preparation method can be applied to the TEM sample detected through electronic Holography detection, and can be widely applied to the obtaining of extrinsic electron holograph electronic Holography information of electric field distribution and magnetic field distribution of the sample.