As an oral vitamin K antagonist, warfarin has been widely used in treating thrombotic diseases for decades. Therefore, the research on the kinetic process of warfarin has become people’s focus. Time-dependent density functional theory (TD-DFT) simulated the excitation and emission spectra and charge transfer processes of several states during the warfarin molecules binding to human serum proteins in aqueous solution. Study the transition mode and charge transfer during the excitation process, the differences in the spectra. Explore the exciting state change mechanism of the entire kinetic process. The results show that the UV-Vis absorption spectrum of warfarin in aqueous solution exhibits double absorption, mainly caused by different excited state transitions. Before deprotonation, the main absorption peak wavelength is 291 nm. After deprotonation, the absorption intensity decreases and the wavelength is red-shifted. When warfarin binds to serum protein, charge transfer occurs, resulting in an absorption gain of 307 nm and the absorption peak intensity is the highest. By calculating the structure and excitation energy of the first excited state (S1) to simulate the fluorescence spectra of different states of warfarin, the fluorescence peak in the initial state is 360 nm. After deprotonation, the fluorescence intensity decreases and the wavelength red shifts. The structural changes after the combination result in fluorescence gain. According to the changes in the fluorescence spectrum before and after the combination of warfarin and protein, warfarin has different fluorescence emission processes in the whole dynamic process. The charge transfer of the entire dynamic process of warfarin molecules is analyzed by molecular frontier orbital and electron-hole methods. The results show that the fluorescence emission process of warfarin monomer is local excitation, and the fluorescence emission process after binding with protein is charge transfer excitation. The combined fluorescence gain feature makes warfarin a fluorescent probe. This paper reveals the mechanism of spectral changes in the warfarin and protein binding process, and provides new research methods and theoretical support for future exploration of molecular binding dynamics.