A calibration method of a detection system including an illumination system configured to illuminate a detection target, and an imaging system configured to form an image of light from the detection target on a photoelectric conversion element, the method including obtaining, for each of at least two combinations of first apertures in the illumination system and second apertures in the imaging system, each of which is formed by selecting one first aperture and one second aperture from the plurality of first apertures and the plurality of second apertures, a defocus characteristic indicating a shift amount of the image on the photoelectric conversion element with respect to a defocus amount of the detection target in a state in which each of the first aperture and the second aperture is positioned in a first position shifted from a reference position.