Abstract: The present disclosure provides a fluorescence image registration method, the method includes: acquiring a fluorescence image of a biochip; selecting a preset local region of the fluorescence image; acquiring a position of a minimum value of a sum of brightness values of pixels in a first direction and a second direction, and obtaining pixel-level registration points; dividing the pixel-level registration points into non-defective pixels and defective pixels; if the fluorescence image meets the preset standard, correcting positions of the defective pixels according to positions of the non-defective pixels; acquiring a position of a center of gravity of image points of fluorescent molecules according to a center of gravity method; fitting straight lines in the first direction and the second direction respectively according to the position of the center of gravity; and acquiring boundary points of the fluorescence image and calculating the positions of the boundary points.