The invention discloses a method for manufacturing a scanning electron microscope sample for an assembled nanometer line array in an aluminum oxide template, which is characterized by comprising the following steps: eroding the template by an alkali liquor the concentration of which is between 0.05 and 5 M, soaking the template continuously in the alkali liquor with lower concentration of between0.001 and 0.0001 M for three to five minutes, then washing the template by water for a few times, and airing the template for the observation under the scanning electron microscope, wherein the alkali liquor is NaOH or KOH solution. The scanning electron microscope sample for the assembled nanometer line array in the aluminum oxide template manufactured by the method can avoid the effect caused by Al(OH)3 deposition attached on the surface of the sample on the observation of the scanning electron microscope.