Scanning electron microscopy (SEM) is widely used to observe graphene on metal substrates. However, the origin of the SEM image contrast of graphene is not well understood. In this work, we performed in situ SEM imaging of layer-number-controlled graphene on a Ni substrate using a high-pass energy filter for secondary electrons. We found that the graphene layer contrast was maximized at 15-20 eV, corresponding to the π-σ* interband transition in graphene. Our results indicate that the SEM image of graphene is produced by attenuation of the electrons emitted from the metal substrate by the monoatomic layers of graphene.