The Seebeck coefficient is an important parameter of thermoelectric materials, which is routinely measured by commercial or home-made equipment based on different methods. However, due to various temperature offsets in the measurement, the determination of temperature gradient can be inaccurate, leading to a large uncertainty in Seebeck coefficient. To elucidate the influence of the inaccurate temperature gradient on the determination of Seebeck coefficient, an error analysis has been performed on a commercial system. Several potential factors that may affect the establishment of temperature gradient were discussed in detail. A comparison between the single point method and the slope method was made to verify which is more accurate to calculate the Seebeck coefficient from the raw measurement data. It is suggested that the slope method is more preferable and the single point method can also be accurate enough when a relatively large temperature gradient is adopted.