Glucose specificity is the premise of spectroscopic measurements of blood glucose concentration, and it is also paramount for feasibility study of a spectral measurement method. Two-dimensional correlation spectroscopy technology is widely used in many fields such as inter-/intra-molecular reaction, material phase transition and information extraction because of its high resolution and effective Noda’s rule. In the present paper, the glucose specificity for noninvasive glucose measurements by mid-infrared spectra based on the 2D correlation spectroscopy was investigated. First, the feasibility of this method was validated by a series of in vitro experiments of glucose. Then the in vivo experiments of four volunteers were conducted and the characteristic information of glucose by mid-infrared spectra collected from human fingers was confirmed by 2D correlation spectroscopy analysis.