A biosensor includes a semiconductor layer having a first surface and a second surface opposite to the first surface, a FET device in the semiconductor layer, an isolation layer over the first surface of the semiconductor layer, a dielectric layer over the isolation layer and the first surface of the semiconductor layer, and a pair of first electrodes and a pair of second electrodes over the dielectric layer and separated from each other. The isolation layer has a rectangular opening substantially aligned with the FET device. The rectangular opening has pair of first sides and a pair of second sides. An extending direction of the pair of first sides is perpendicular to an extending direction of the pair of second sides. The pair of first electrodes is disposed over the pair of first sides, and the pair of second electrodes is disposed over the pair of second sides.