We report the 532 nm-excited hyper-Raman (HR) spectrum of D-glucose in phosphate-buffered saline (PBS) at pH 7.4. Large and unique HR bands were observed from 1500 to 1650 cm−1. The HR spectral pattern was completely different from those of infrared absorption and Raman spectra. The HR bands were affected by isotope labeling on glucose but not phosphate. The HR bands were attributed to the combination or overtone modes of glucose. These bands were also observed in buffers containing phosphate, carbonate, and Tris but not in H2O. We tentatively attributed the origin of the HR bands to the charges of the buffer molecule(s) and the resultant electric field effect on the electronic and/or vibrational wavefunctions.