Disclosed is a positioning method for biochip spotting, which can spot the bio-probe precisely on the specific position of a biochip substrate, and the steps thereof comprising: (a) providing reference points on a biochip substrate; (b) taking an image of the position of each reference point; (c) calculating a first spotting position and performing spotting according to position information of the image of the positions of reference points of step (b); (d) taking and recording the image of the spotted position; (e) calculating to make feedback correction and setting up the next spotting position according to the position information of the image of step (d) and then performing spotting; and (f) repeatedly performing step (d) to step (e) until all spotting is completed.