The invention relates to the field of experiment equipment, in particular to a test tube cleaning device. The test tube cleaning device comprises a base, a first air cylinder, a stepping motor, an air cylinder fixing frame, a second air cylinder, a water storage tank, water injecting ports, a brush rod, water spraying ports and a brush head, wherein the first air cylinder and the base are fixed together in a welding mode, the first air cylinder is installed on the upper portion of base, the stepping motor is installed above a piston rod of the first air cylinder, the second air cylinder is installed above a rotary shaft of the stepping motor, the water storage tank is installed at one end of a piston rod of the second air cylinder, the water injecting ports are formed in the upper surface of the water storage tank, the brush rod is installed on the lateral surface of the water storage tank, the brush head is installed at one end of the brush rod, and the water spraying ports are in the periphery of the brush head. The test tube cleaning device can assist in cleaning test tubes.