The invention discloses a high-precision axial positioning and imaging method for an ultra-thin cell. The upper surface and the lower surface of the cell are divided into two parts; fluorescence light intensity information corresponding to the axial position is obtained through multi-angle illumination of different modes on the two parts; and axial information of fluorescent particles in the cell is obtained by combining a corresponding algorithm to obtain a three-dimensional image. The invention further discloses a high-precision axial positioning and imaging device for the ultra-thin cell. Circular scanning is carried out on illuminating light on the surface of a sample through a 1/4 wave plate and a two-dimensional scanning galvanometer system and the incident angle is changed at the same time to generate two different illumination modes; excited fluorescent light is received by a CCD; and high-precision axial positioning of the ultra-thin cell can be achieved by combining the relationship between the received fluorescent light intensity and the axial position of the sample. The device is simple, and convenient to operate; and high-precision positioning and imaging are carried out on the upper and the lower part of the whole cell by using different incident angles of the excited light.