In vitro models of the liver have a good simulation of the micro-liquid environment inside the human liver and the communication between cell tissues, which provides an important research tool for drug research and liver disease treatment. In this paper, we designed a 3D liver chip and real-time monitoring system based on microfluidic technology. The in vitro model of the liver on the chip was established by the three-dimensional microfluidic chip pipeline and the corresponding microwell array. Meanwhile, the culture medium is continuously injected on the chip, and the electrochemical impedance spectroscopy and near-infrared spectroscopy of the liver chip are recorded and analyzed from day one to day five. When the 3D cultured liver chip in vitro model reached a certain period and stabilized, paracetamol with varying gradients of concentration was applied to the cultured cells for drug resistance testing. The experimental results show that the liver chip and its monitoring system designed in this paper can maintain 100% cell viability of hepatocytes in vitro for a long time. Furthermore, it can meet the requirements of measurement technologies such as electrical impedance measurement and near-infrared spectroscopy in real-time, providing a stable culture platform for the further study of organ chips.