Lyot filter is widely used in solar observation for spectra-scanning imaging. Calibration experiment at regular intervals is an important work to assure the accuracy and validity of Lyot filter. This paper comes up with a new method to conduct the Lyot filter calibration experiment on-line while traditional method requires perfect stability of environment. This method uses monochromatic imaging channel and Lyot filter scanning imaging channel simultaneously, and corrects the scanning data with monochromatic imaging data to correct the impact of environment. The instability of light source caused by disturbance of observation environment is reduced. We apply the calibration method in the high-resolution multi-wavelength solar imaging system to calibrate the Lyot filter in Hɑ (656.28) scanning imaging channel and correct the scanning data with TiO band (705 nm) observation data. The result shows that this method successfully eliminate the impact of the light instability on scanning curve of Lyot filter. The difference between the ideal center and the true center of the filter is more than 0.005 nm. The accuracy of the calibration experiment and the adaptability to environment are promoted.