A novel approach to calibrate the sensitivity of a differential thermometer, consisting of several thermocouples connected in series (thermopile), has been developed. The goal of this method is to increase the accuracy of small temperature difference measurements (∆T ≤ 1 K), without invoking higher sensor complexity. To this end, a method to determine the optimal temperature difference employed during the differential measurement of thermoelectric sensitivities has been developed. This calibration temperature difference is found at the minimum of combined measurement and linearization error for a given mean temperature. The developed procedure is demonstrated in an illustrative example calibration of a nine-junction thermopile. For mean temperatures between −10 • C and 15 • C, the thermoelectric sensitivity was measured with an uncertainty of less than ±2 %. Subsequently, temperature differences as low as 0.01 K can be resolved, while the thermometer used for the example calibration was accurate only to ±0.3 K. This and higher degrees of accuracy are required in certain research applications, for example to detect heat flux modulations in bifurcating fluidic systems.