A manufacturing method of an image sensor is provided. A substrate is provided, and the substrate includes a pixel array region. A plurality of openings is formed in the pixel array region of the substrate. A light guide region is formed in the substrate aside each of the openings, wherein a portion of the substrate is disposed between the light guide region and the opening, and the depth of the light guide region in the substrate is greater than the depth of the opening aside the light guide region in the substrate. Isolation structures are formed in the openings to define a plurality of pixel regions respectively located between two adjacent isolation structures in the pixel array region. A photosensitive region is formed in each of the pixel regions of the substrate. A conductive line layer is formed on each of the pixel regions of the substrate.