The invention provides a calibration method for reflective machine vision illumination systems. The illumination system comprises a graphic data generation device capable of generating graphic data ofillumination required by machine vision; an optical signal output device capable of outputting an optical signal of illumination required for machine vision according to the graphic data; an opticalpath adjusting device capable of adjusting an optical path of the optical signal such that the optical signal provides illumination to a subject in a desired manner; the optical path adjusting devicecomprises a plurality of optical path adjusting units; the graphic data comprises graphic units corresponding to each optical path adjusting unit. The method comprises the following steps of: aligningeach optical path adjusting unit with a corresponding graphic unit; brightness calibration step; color temperature calibration step.