The variation of temperature is one of the main interference factors that affect the accuracy of near-infrared (NIR) non-invasive blood glucose sensing. In order to reduce the influence of temperature variation on NIR diffuse reflectance, a measurement method based on temperature insensitive source-detector separation was proposed in this paper, i. e., the temperature insensitive source-detector separation, whose diffuse reflectance was insensitive to temperature variations, was chosen to obtain the spectrum. The Monte Carlo method was used to simulate the diffuse reflectance at multiple source-detector separations with temperatures of 30～40 ℃ and glucose concentrations of 0～300 mmol·L-1. According to the simulation results, the existence of temperature insensitive source-detector separation in human skin tissue model and the influence of glucose concentration on the separation were analyzed. At wavelength of 1 000 nm, the correlation between the diffuse reflectance and glucose concentration at different source-detector separations under random temperature and constant temperature were compared. Furthermore, the partial least square (PLS) model of glucose were established using the diffuse reflectance at temperature insensitive source-detector separations and other separations under six wavelengths (1 000, 1 050, 1 100, 1 150, 1 350 and 1 410 nm), and the prediction accuracy of the model under random temperature and constant temperature were compared. The results showed that, in the range of 1 000～1 440 nm, there were temperature insensitive source-detector separations in human tissue, and the influence of glucose concentration variationon it can be ignored. When the temperature of the sample variates, both the correlation between glucose concentrations and diffuse reflectance and the prediction accuracy of model obtained at the temperature insensitive source-detector separation were significantly better than that of other separations, which was almost close to the situation of constant temperature. Therefore, the measurement method based on the temperature insensitive source-detector separation can effectively reduce the influence of temperature variations on NIR diffuse reflectance, and it is expected to improve the accuracy of NIR diffuse reflectance in non-invasive blood glucose sensing.