The present invention discloses an image sensor for real-time calibration of dark current, including a pixel array comprises at least a pixel unit, the pixel unit includes a pixel photosensitive portion, a pixel dark shielding portion and a subtraction circuit, photodiodes in the pixel photosensitive portion and the pixel dark shielding portion are isolated by deep trench isolations, the pixel dark shielding portion are covered by a dark shielding layer; both of the pixel photosensitive portion and the pixel dark shielding portion adopt a same voltage and sequential control, a light ambient voltage signal and a non-light ambient voltage signal are generated and connected to both ends of a subtraction circuit to realize subtraction and dark current calibration. The present invention discloses an image sensor for real-time calibration of dark current, which can make the dark current calibration completed directly within the pixel, and can better cover the dark pixel part, so as to make calibration value of dark current and dark noise more accurate.