Measuring the glucose concentrations in the interstitial fluid is currently the main method to achieve the continuous blood glucose monitoring. The MIR-ATR(Mid-infrared, Attenuated Total Reflection)Spectroscopy has prominent advantage on the analysis of small biological molecule for composition information like the glucose, but it is still an unresolved problem that how to detect the subcutaneous glucose concentration by using the MIR-ATR Spectroscopy. In the present paper, we carry out the experiment based on MIR-ATR for the detection of subcutaneous glucose information on both the natural state and the penetration state based on the theoryanalysis of MIR penetration depth. Firstly, collect spectral data of the subcutaneous glucose concentration of human finger on the natural state were collected as the light shined the skin directly, and it was discussed whether the MIR can penetrate the skin to get the information of subcutaneous glucose. On this basis, collect spectral data of the subcutaneous glucose concentration of human finger at the penetration state were collected when the Interstitial fluid is permeated to the surface layer by using low-frequency ultrasound and vacuum, then it analyzed that whether it can detect the glucose-specific information or not. As the two-dimensional correlation spectroscopy has high resolution and good versatility, it is widely used to analyze the inter-molecular reaction and judge the absorption peaks information in many fields including the MIR spectroscopy field, so we choose the Two-dimensional correlation spectroscopy to analyze the information of subcutaneous glucose concentration at the natural state and the penetration state. The experiment result shows that the MIR-ATR spectroscopy can’t be applied in the detection of subcutaneous glucose concentrationdirectly, and it is a promising direction to make the Interstitial fluid permeated to the surface layer by the physical methods or chemical methods.