Volumetric imaging of a mouse brain in vivo with one-photon and two-photon ultralong anti-diffracting (UAD) beam illumination was performed. The three-dimensional (3D) structure of blood vessels in a mouse brain was mapped to a two-dimensional (2D) image. The speed of volumetric imaging was significantly improved due to the long focal length of the UAD beam. Comparing one-photon and two-photon UAD beam volumetric imaging, we found that the im-aging depth of two-photon volumetric imaging (80 μm) is better than that of one-photon volumetric imaging (60 μm), and the signal-to-background ratio (SBR) of two-photon volumetric imaging is two times that of one-photon volumetric imaging. Therefore, we used two-photon UAD volumetric imaging to perform dynamic volumetric imaging of mouse brain blood vessels in vivo and obtained the blood flow velocity.