The invention belongs to a seedling separating device of a pot seedling transplanting machine in the field of automatic transplanting in facility agriculture. The seedling separating device comprises a mounting rack, a linear transmission device, a supporting cup opening and closing device, a supporting cup moving device, a plurality of supporting cups and a control system, wherein the linear transmission device is fixedly arranged on the mounting rack; the supporting cup opening and closing device is mounted on the mounting rack and arranged behind a cup holder, the supporting cup opening and closing device drives the lower part of the cup holder to be opened in a forward stroke and closed in a return stroke; the supporting cup moving device is fixedly arranged on the linear transmission device; the supporting cups are arranged on the supporting cup moving device; the linear transmission device drives the supporting cups to arrive at a specified location and receive pot seedlings grabbed by a seedling dropping claw. The seedling separating device of the pot seedling transplanting machine is simple in structure, controllable in procedure and high in reliability; a motor drives the supporting cups to move left and right, a cylinder pushes the supporting cups to be opened and closed, the supporting cups are vertically downward, seedling dropping posture is better, and location of a seedling dropping point also can be regulated; accurate seedling distribution at the specified location and a seedling separating action that the seedlings are dropped from lower openings of the supporting cups in two rows can be realized.