Heterogeneously integrated and miniaturized neural sensing microsystems are crucial for brain function investigation. In this paper, a 2.5D heterogeneously integrated bio-sensing microsystem with μ-probes and embedded through-silicon-via (TSVs) is presented for high-density neural sensing applications. This microsystem is composed of μ-probes with embedded TSVs, 4 dies and a silicon interposer. For capturing 16-channel neural signals, a 24 × 24 μ-probe array with embedded TSVs is fabricated on a 5×5 mm(2) chip and bonded on the back side of the interposer. Thus, each channel contains 6 × 6 μ -probes with embedded TSVs. Additionally, the 4 dies are bonded on the front side of the interposer and designed for biopotential acquisition, feature extraction and classification via low-power analog front-end (AFE) circuits, area-power-efficient analog-to-digital converters (ADCs), configurable discrete wavelet transforms (DWTs), filters, and a MCU. An on-interposer bus ( μ-SPI) is designed for transferring data on the interposer. Finally, the successful in-vivo test demonstrated the proposed 2.5D heterogeneously integrated bio-sensing microsystem. The overall power of this microsystem is only 676.3 μW for 16-channel neural sensing.