In biomedical photonics, it′s extraordinarily significant to diagnose lesion by quantificational optical parameters. The high resolution absorption and refractive index spectra of samples with different mass fractions of anhydrous glucose are acquired by terahertz time-domain spectroscopy system in nitrogen atmosphere at room temperature. The characteristic absorption is analyzed based on the density functional theory. Gaussian 09 is used to simulate the optimized structure and infrared spectroscopy of single, double, triple and quadruple glucose molecules. Meanwhile, the B3LYP method and 6-31G basis set are adopted. In addition, Materials Studio 7.0 is utilized to calculate the frequency generated by the glucose crystal structure, which is completed with the help of CASTEP module and the generalized gradient approximation method. The experimental results show that the absorption peaks of anhydrous glucose appear at 0.94, 1.30, 1.44, 1.67, 1.88, 2.08, 2.31, 2.55, 2.70, 2.84, 2.96 THz, and the intensities of all peaks decrease linearly with the increase of glucose mass fraction in samples. The results of Gaussian 09 are in agreement with the test results, which is verified by the results of CASTEP. The absorption peaks of anhydrous glucose are identified accurately in the range of terahertz waveband. The reuslts provide experimental and theoretical reference for the application of terahertz time-domain spectroscopy in biomedicine.