As one of the major causes of cancer-related death, gastrointestinal cancer seriously impacts the quality of human life. Moreover, in the majority of patients, the disease is in an advanced state at the time of diagnosis. The only available treatment for such patients is systemic chemotherapy, which may cause various side effects. Thus, the need for fast evaluation functions of different medicines and developing new drugs have become urgent in recent years. In this study, four different anti-cancer reagents, namely, compound Kushen injection (CKI), cinobufacini injection, transforming growth factor-β1 (TGF-β1), and 5-fluorouracil (5-FU), were each tested with a cell-based biosensor to investigate their therapeutic effects on gastrointestinal cancer cells. From the impedance results, we could infer that chemotherapeutic agents could induce cell apoptosis, disrupt cell cycle, and inhibit cell adhesion capacity. By combining impedance spectra with an appropriate impedance circuit, the function of 5-FU on cell death and adhesion could be preliminarily investigated. Because the impedance sensing is in real time, has high throughput, and is easily manipulated, the developed cell-based impedance biosensor could be used as a suitable and promising alternative to end-point assay in the preclinical research of medicines, including the development of new therapeutic regimens.