An experimental and digital procesing method for eliminating the influences of the autocorrelation item on the reconstructed image of an electron hologram by use of a spatial subtraction was described.In this method,two images were involved.One was recorded when a high voltage was put on the electron biprism and another was recorded after the voltage of the electron biprism returned to zero.For eliminating the energy and location deviation between the two-recorded patterns an iterative algorithm was adopted,in which the total energy of the zero diffraction order was taken as the criterion.The experimental results verified the feasibility of this method.