An image sensor includes a pixel unit and a pixel readout circuit. The pixel unit includes an image pixel array including a plurality of image pixel columns, respectively; a first reference pixel array including a plurality of first reference pixel columns; and a bias circuit, coupled to the image pixel columns for generating a plurality of column sensing signals, coupled to the first reference pixel columns for generating a plurality of first reference signals, and further for generating a first average reference voltage signal according to the plurality of first reference signals. The pixel readout circuit generates a plurality of reset values and a plurality of sampling values according to the column sensing signals and the first average reference voltage signal, wherein the plurality of reference pixel rows is less than the plurality of image pixel rows.