For measurement of substance concentration in turbid medium by near-infrared diffuse spectroscopy, the measurement sensitivity and the position of the adopted spectrum detector, i.e. source-detector separation (SDS), are closely related. By analyzing the sensitivity of substance concentration measurement curved with changes of SDS, three special SDSs with maximum, minimum (zero) and local maximum sensitivities were obtained, respectively. In the glucose measurement experiment in turbid medium solution, Intralipid solution with water, glucose, hemoglobin and albumin, the three special positions of SDS were verified by diffusion equation, Monte Carlo simulation and diffuse reflection spectroscopy. Considering the limiting factors such as stability of light source, detective quantum efficiency, noises of InGaAs detector, we used the signal-noise ratio (SNR) as another indicator to evaluate the sensibility of an actual measurement. After comprehensively analyzing the sensibility graphs and actual measurement conditions, we presented the optimized SDS which the glucose measurement precision was improved by more than 10 times. The method for improving glucose measurement sensitivity in turbid medium could be referenced and practiced in non-invasive blood glucose measurement or other substance concentration measurement using near-infrared spectroscopy.