A biopsy needle with electrical impedance sensor array based on stainless steel microelectrodes (EIS needle) was developed for real-time four electrode measurement and multi-spot sensing of tissues during the biopsy process. The sensor performance was characterized by using saline solutions with various concentrations, which proved accurate, stable and reliable electrical impedance measurement. The capability of impedance-based tissue sensing was verified by the conductivity measurement of agarose hydrogel based phantom mimicking cancer tissue. Furthermore, multi-spot impedance sensing during needle insertion was demonstrated using porcine meat with muscle and fat layers, which exhibited a clear discrimination between different types of tissues. Also, the electrical impedance difference between normal and fatty livers of mouse model was measured by the EIS needle. We could successfully demonstrate that the EIS needle can provide localized and accurate characterization of biological tissues at the needle tip.