In the traditional neural sensing microstructure, the limited metal line pitch and the metal layer numbers restrict the neural signal routing ability from electrodes to circuit chips. Miniature packaging and excessive noise interference bottlenecks are some of the challenges faced by the electrodes and circuit chips integration with traditional wire bonding. This paper proposes a 2.5-D heterogeneous integration neural sensing microsystem based on the silicon substrate to overcome these issues. With standard semiconductor and 3-D integration processes, high-channel-density (256 channels at 25 mm2) neural sensing microsystem is achieved. Through silicon via provides the shortest vertical interconnection and dramatically minimizes the packaging. Furthermore, the interposer can carry multiple chips to enhance the function of the biosensor. Electrical characteristics and reliability examinations reveal its high quality and great performance as compared to traditional approaches. This novel highly integrated neural sensing microsystem is expected to contribute to the biomedical field for exploring and solving unknown biological mysteries.