An image sensor and a manufacturing method thereof are provided. The image sensor includes a pixel sensing circuit, a pixel electrode, and an opto-electrical conversion layer. The pixel sensing circuit is corresponding to a plurality of pixel regions. The pixel electrode is disposed on the pixel sensing circuit. The pixel electrode includes a first electrode and a second electrode and is electrically connected to the pixel sensing circuit. The first electrode and the second electrode are coplanar, and have different polarities. The opto-electrical conversion layer is disposed on the pixel sensing circuit. The opto-electrical conversion layer includes a plurality of opto-electrical conversion portions, each of the opto-electrical conversion portions is corresponding to each of the pixel regions, and the opto-electrical conversion portions are separated from each other by a pixel isolation trench.