In order to verify the theoretical relationship of the diffraction efficiency of multi-layer diffractive optical elements varying with the angle of incidence, an optical system with multi-layer diffractive optical elements is designed and manufactured. To ensure the measurement accuracy of diffraction efficiency when light of secondary order diffraction is received by the detector after passing through the pinhole aperture, a correction method of first-order diffraction energy is proposed. The diffraction efficiency of the multi-layer diffractive optical elements with the incident angles ranging from 0° to 30.6° is measured by the established double light-path measurement device. Diffraction efficiency is measured at seven different incident angles with the designed wavelength of 532 nm, and the measurement results are simulated and analyzed. Due to some manufacturing errors, the diffraction efficiency obtained by the actual measurement is lower than the theoretical analysis results. However, both measured and theoretical results show that the diffraction efficiency of multi-layer diffractive optical elements declines with the increase of incident angle.