The invention discloses a light slice fluorescence microscopic imaging method and device based on relocation, and belongs to the technical field of optical imaging, the light slice fluorescence microscopic imaging device comprises a laser, a cylindrical mirror, a sample stage carrying a fluorescent sample, and a detection system collecting fluorescence emitted by the fluorescent sample, the laser,the cylindrical mirror and the sample stage arranged along the optical path in turn, the detection system includes a first detector, a beam splitter, a second detector, and a third detector, the detection system also includes a processor coupled to the detection system and the sample stage, the processor controls the sample stage to move along Z-axis in a fixed step length, compares a fluorescence image I1 and a fluorescence image I2 to obtain position information of each part of the fluorescence on the Z-axis, and the fluorescence information in a fluorescence image I0 is repositioned and three-dimensionally reconstructed according to the position information to obtain a three-dimensional imaging result of the fluorescent sample. The axial resolution of imaging can be improved without reducing the range of an imaging view field, increasing photobleaching of the sample, and reducing of imaging speed.