Based on strain gradient theory, a characterization method to describe the constitutive relationship of an aluminum grating with the glass substrate is proposed. A mathematical model is established including the constitutive relationship of the glass substrate and the aluminum film parameters and the corresponding parameters are characterized. In order to verify the accuracy of the proposed constitutive relationship, the nano-indentation experiment of the 79 g/mm echelle aluminum grating is carried out. It proposes an assumption that the grating film and substrate materials have the size effect, then performs a nano-indentation experiment to verify the size effect and the substrate effect for the aluminum film. As compared with grating aluminium film experiments, the results on the nano-indentation under the conditions with and without substrates show the differences for 0.8 times in the direction of stress and 3 times in the direction of strain. A grating ruling experiment is also carried out, and the results indicate that the indentation experiment has an important guiding role for the ruling experiment. The research process and results show that theoretical analysis and two kinds of experiments can effectively help to analyze the grating ruling process and reduce the errors of grating ruling. The method provides a good theoretical guidance for the process of the grating ruling.