Circular dichroism (CD) spectroscopy is a well-established biophysical technique used to measure protein and its secondary structure and to detect changes in secondary and higher orders of structure for applications in research and the quality control of protein products such as biopharmaceuticals. However, objective comparison of spectra is challenging because of a limited quantitative understanding of the sources of error in the measurement. Statistical methods can be used for comparisons but do not provide a mechanism for dealing with systematic and random, errors. CD measurements in any two instruments may often present slight differences in spectral magnitude or wavelength, even for the same sample under comparable conditions. The small disparities between the polarization of the incident light from each instrument, light source, and final lamp output are examples of the variables that can produce such differences. On the other hand, the structural information acquired with the CD method can sometimes be hampered by the poor quality of the original CD data, and CD deconvolution analysis strongly depends on the spectral intensity. Here a helix predominate protein—cytochrome C was taken as the experimental object, and CD spectroscopy was used to measure the concentration of 0.05 mg·mL-1 cytochrome C aqueous solution after instruments were typically calibrated using standards. And then, a measurement model for CD spectroscopy of 0.05 mg·mL-1 Cytochrome C aqueous solution was presented, incorporating the principal sources of uncertainty to derive an uncertainty budget of spectral magnitude in wavelength 222 nm. The uncertainties of spectral magnitude were from measurement repeatability, concentration uncertainty of calibration solution and protein solution, the uncertainty of cell length of the cuvette, etc. After calibrating the instrument, these sources of uncertainty were comprehensively considered, and the magnitude uncertainty of 0.05 mg·mL-1 cytochrome C aqueous solution at the wavelength of 222 nm was (-4.53±0.54) mdeg, k=2. The uncertainty, evaluation found that the uncertainty of 1 mm cuvette cell length and the solution preparation process account for a significant part of the uncertainty component. Eliminating or reducing the impact of these factors can improve the measurement method to analyze the measurement process to achieve an objective comparison of CD spectra and improve the comparability and reliability of CD spectra. This work also provides an experimental reference for the interlaboratory comparison of circular dichroism measurement.