The invention discloses an in-situ sampling apparatus for epilithic periphyton. According to the invention, the apparatus fixes a sampling cross-sectional area and uses an eccentric nylon brush which rotates at a high speed under the driving of a motor to brush off epilithic periphyton, wherein the brush is arranged on a probe; negative pressure is formed in a bowl-shaped sealing rubber ring positioned at the end of the probe and in the probe body under the action of a vacuum pump so as to allow water to enter into the probe along a slit between a stone and the sealing rubber ring, so it is guaranteed that periphyton is not taken away by a torrent when brushed off; and periphyton suspension is sucked into a sample collection bottle through a sample suction hole in the probe and deposited at a cone bottom, and a periphyton sample is separated through a sample separation valve. The in-situ sampling apparatus provided by the invention has the advantages of simple operation, high sample fidelity and a fast sampling speed and overcomes the problem that in-situ collection of epilithic periphyton on a large stone cannot be realized in a torrent. The in-situ sampling apparatus is applicable to a sample region where heterotopic manual sampling can be carried out.