In the studies of fragmentation processes of molecules induced by extreme ultraviolet photons, intense laser fields, or charged particles, kinetic energy release (KER) is a key physical parameter. It can reveal the electronic states of the parent molecular ion, and provide an insight into the molecular structures and the dissociation dynamics. Therefore, it is essential to obtain the accurate KER spectrum for studying the fragmentation process of molecules. However, in the experiments using reaction microscope, experimental parameters such as the time-of-flight (TOF), the voltage of the TOF spectrometer and the detector image of the fragments have significant influence on the accuracy of KER determination. In this work, by taking the two-body fragmentation process of CO ²⁺→ C ⁺+ O ⁺induced by 108 keV/u Ne ⁸⁺impact on CO molecules as a prototype, we introduce two methods to accurately calibrate the reconstructed KER spectrum. The first method is to employ two-dimensional momentum spectra of C ⁺ions obtained by slicing the momentum sphere. The parameters are correctly calibrated when the circular distribution of the two-dimensional ion momentum image is restored. The second method is to use the correlation spectra of the KER as a function of the emission angle of the C ⁺ions to calibrate the experimental parameters, the calibration meets the required level only when the linear dependence of the emission angle on the KER is fulfilled. Then, calibrated KER spectrum is obtained for the dissociation process. By fitting the peak dissociated from the $^{3}\Sigma^{+}$ state of CO ²⁺in the KER spectrum, the energy resolution is estimated at 0.24 eV under these experimental conditions. Although these two methods can be used to accurately calibrate the reconstructed KER spectrum, the second calibration method does not require particularly high data statistics, and is suitable for analyzing the processes with lower reaction cross section. Furthermore, this method is convenient for debugging the parameters. Both methods are reliable for parameter calibration and guarantee high accuracy KER for molecular fragmentation experiments in future.