Abstract: The present disclosure belongs to the technical field of amniotic membrane, and in particular, relates to a porous biological amniotic membrane puncture device and a preparation method. The device includes a sliding table, a light-shielding frame, a base I, and a base II. The sliding table is provided with a first sliding groove; the light-shielding frame is slidably connected to the first sliding groove; a three-axis movable platform is disposed on the base I; the three-axis movable platform is fixedly connected to a laser puncture device; an upper end of the base II is fixedly connected to a fixed board surface; a middle portion of the fixed board surface is provided with an embedded groove; and an amniotic membrane sample is placed in the embedded groove.