A biosensor device includes a substrate, a sensing transistor, an isolation layer and a main interface layer. The sensing transistor is formed on the substrate and including a bottom gate structure, a top gate structure and a semiconductor layer disposed between the bottom gate structure and the top gate structure. The bottom gate structure is electrically connected to the top gate structure. The isolation layer is formed on the sensing transistor for covering the sensing transistor, and includes a first opening. The main interface layer is disposed in the first opening for receiving biomolecules to be sensed. The main interface layer is electrically connected to the top gate structure.