In order to improve the accuracy of near-infrared (NIR) spectroscopy measurement, the method with medium concentration sample as reference for relative measurement is proposed. Firstly, theoretical analysis of absorbance error sources with background reducted is presented, which shows that errors caused by sample cell and optical fiber loss can be reduced by using medium concentration sample as reference. Furthermore, NIR transmission experiments on glucose aqueous solution and 3% intralipid solution as phantom are performed with glucose used as analyte. In the double-beam experiment on glucose aqueous solution, root mean square error of calibration (f_RMSEC) and root mean square error of cross validation (f_RMSECV) of the partial least squares (PLS) regression model using medium concentration sample as reference decrease respectively by 36.6% and 41.0% compared to the model with pure water as reference. Meanwhile, in the intralipid phantom experiment, fRMSEC and fRMSECV of the PLS regression model using medium concentration sample as reference decrease respectively by 66.3% and 39.3% compared to the model with glucose- free 3% intralipid solution as reference. The results show that the accuracy of NIR transmission spectroscopy analysis can be effectively improved by using medium concentration sample as reference.concentration sample