Fluorescence spectroscopy provides a noninvasive method to quantify biochemical and structural changes in tissues and it is regarded as an early subtle indicator of disease or injury.One challenge facing the rapid growth of fluorescence application is the inability to insure the accuracy of fluorescence measurements.The calibration protocols that include excitation energy calibration,collection efficiency calibration,wavelength calibration,system nonlinearity calibration,system response calibration,background subtraction and spectrum signal filtering,for optical instruments that ensure measurement accuracy are proposed.Three fluorescence spectroscopy systems are built and the fluorescence spectra of fluorescence standard and human skin are collected.The result shows that the spectrum intensity variation coefficient among these systems for the same reference standard is 7.4% and for the same human skin is 10.4% before calibration.By comparison,the spectrum intensity variation coefficients after calibration are 0.9% and 2.0%,respectively.