An object of the present invention is to provide a substrate with a multilayer reflective film that enables the number of detected pseudo defects, to be reduced even when using highly sensitive defect inspection apparatuses using light of various wavelengths, and in particular, is capable of achieving a level of smoothness required of substrates with a multilayer reflective film while reliably detecting critical defects as a result of reducing the number of detected pseudo defects, as well as a method of manufacturing the same.The present invention relates to a method of manufacturing a substrate with a multilayer reflective film having a multilayer reflective film, obtained by alternately laminating a high refractive index layer and a low refractive index layer, on the main surface of a mask blank substrate on the side of which a transfer pattern is formed, comprising a step of: depositing the multilayer reflective film on the main surface by ion beam sputtering using targets composed of a high refractive index material and a low refractive index material; wherein, during the ion beam sputtering, sputtered particles of the high refractive index material and the low refractive index material are made to enter at prescribed incident angle relative to the normal of the main surface so that the power spectral density in a prescribed spatial frequency region is a prescribed value.