The unburned carbon in fly ash is an important indicator of the coal-fired boiler combustion efficiency. Laser-induced breakdown spectroscopy (LIBS) has been applied for measurement of unburned carbon for its fast response. Traditionally, the unburned carbon content determined by the loss-on-ignition (LOI) method was used as the reference value of the LIBS calibration model. However, the reference value of unburned carbon content would not correspond to the LIBS spectral information because the result measured by the LOI method represents not only unburned carbon but also decomposition of some mineral phases in fly ash during ignition. In this paper, different values of unburned carbon content determined by the loss-on-ignition method and elemental analysis technique, respectively, were compared as the reference of LIBS calibration model. The analysis results showed that there is a better correspondence between the total carbon content determined by elemental analysis technique and the LIBS spectral information on fly ash. Compared with the reference value determined by the LOI method, the regression coefficient (R-2) of the calibration model by the reference value determined by elemental analysis technique improved from 0.980 to 0.992, and the root-mean-square error of prediction decreased from 0.702% to 0.111%. This research demonstrates that the total carbon content is more suitable to be used as reference value to establish a calibration model of unburned carbon content in fly ash by LIBS analysis.