This paper presents the fabrication and characterization of a three-dimensional (3D) thermopile; consists of 71 Cu–Ni series-connected thermocouples on polyimide (PI) flexible substrate. Using wet etching to etch through 25 μm PI, the cold and hot junctions of thermocouples are formed on the top and bottom surfaces of PI substrate. This 3D layout design differentiates its innovative uniqueness from the traditional 2D planar thermopiles that have both hot and cold junctions on the same plane. The experimental studies on the PI etching with respect to the concentrations of KOH and C2H7NO in the etching solution conclude that the optimal composition of the etchant is 7–9 M KOH with 2–4 M C2H7NO and etched at 80 °C. A measured sensitivity of 0.44 mV/K is realized in the fabricated device. The temperatures measured by the 3D thermopile are very close to those obtained with a digital thermometer, demonstrating that 3D flexible thermopiles has great potential to provide low cost thermal sensor.