The invention discloses a preparation method of a scanning electron microscope sample. The preparation method comprises the following steps: treating a feces sample, wrapping and washing the feces sample in a nylon bag, standing and washing the feces sample in a culture dish, flushing by using a high-speed water flow, changing water, drying by absorbing water, preparing a sample platform, and coating the sample with a film by gold sputtering. The scanning electron microscope sample prepared by drying, crushing, soaking, wrapping and washing in the nylon bag, flushing by using the high-speed water flow, absorbing water, drying, plating gold and other processes is clean, free of impurities and gravel deposits, small in residue loss and uniform in distribution; during macroscopic observation, the residue sample is clear, and during observation under a scanning electron microscope, a residue structure is clear and complete, the view is clean and the third dimension of an image is strong; by the preparation method, observation and research on ultrastructure of an undigested forage grass residue in digested feces of a ruminant animal can be facilitated; the preparation method has an important significance in vividly understanding and evaluating degradation of microbes in the digestive tract of the ruminant animal to a roughage fiber structure and raising the utilization rate of straw feed.