In vivo sensing of various physical/chemical parameters is gaining increased attention for early prediction and management of various diseases. However, there are major limitations on the fabrication method of multiparameter needle-based in vivo sensing devices, particularly concerning the uniformity between sensors. To address these challenges, we developed a microscale biosensor array for the measurement of electrical conductivity, pH, glucose, and lactate concentrations on a flexible polymeric polyimide platform with electrodeposited electrochemically active layers. The biosensor array was then transferred to a medical needle toward multiparametric in vivo sensing. The flexibility of the sensor platform allowed an easy integration to the curved surface (φ = 1.2 mm) of the needle. Furthermore, the electrodeposition process was used to localize various active materials for corresponding electrochemical sensors on the microscale electrodes with a high precision (patterning area = 150 μm × 2 mm). The biosensor array-modified needle was aimed to discriminate cancer from normal tissues by providing real-time discrimination of glucose, lactate concentration, pH, and electrical conductivity changes associated with the cancer-specific metabolic processes. The sensor performance was thus evaluated using solution samples, covering the physiological concentrations for cancer discrimination. Finally, the possibility of in vivo electrochemical biosensing during needle insertion was confirmed by utilizing the needle in a hydrogel phantom that mimicked the normal and cancer microenvironments.