The progress of non-invasive blood glucose determination based on mid-infrared spectroscopy is introduced firstly. After this, the effect of stratum corneum (SC) on blood glucose determination is studied by experiment while interstitial fluid (ISF) is collected on the skin surface. Using the spectra of simulated ISF, partial least square regression (PLSR) calibration model of glucose determination is estabished, and the value of root mean squares error of cross validation (RMSECV) is 9.6 mg/dL. While including the interference of stratum corneum from three volenteers individually, three RMSECV are obtained, and they are 17.4, 16.3 and 17.1 mg/dL, respectively. The results show that the effects of SC from different volenteers are the same nearly; including the interference of SC, the accuracy of the non-invasive blood glucose determination based on attenuated total reflection mid-infrared spectroscopy can reach 18 mg/dL.