The application of tunable Mid-infrared laser system based on fiber-optic ATR sensor to regent-free quantification of glucose concentration was presented. The five laser emission wavelengths, including 1081, 1076, 1051, 1041 and 1037 cm-1, were employed for glucose determination. In our experiments, absorbance at the five wavelengths correlates strongly well with glucose concentration (R2>0.99, SD<0.0004, P<0.0001), and the noise-equivalent concentration is as low as 3.8 mg/dL. Compared with the conventional FT-IR spectrometer, higher sensitivity was aquired because of the laser higher power and spectral resolution, and it is about 4 times as high as that of FT-IR spectrometer. All the results of this investigation suggested that the tunable CO2 laser spectroscopy is a powerful method for glucose measurement. Especially, the multiple tunable wavelengths, which makes it possible for glucose determination in blood or interstitial fluide with complicated components.