Revealing the binding properties of calcium ion (Ca2+) and magnesium ion (Mg2+) to chromophoric dissolved organic matter (CDOM) facilities understanding the effect of natural water composition on the photophysics of dissolved organic matter. Steady-state and time-resolved fluorescence spectrometry, and dynamic light scattering were applied to investigate the fluorescence quenching process of CODM by Ca2+ and Mg2+. The binding of Ca2+ and Mg2+ preferred terrestrial CDOM to aquatic CDOM. The fluorescence quenching of CDOM by cations mainly occurred in a static process, which was based on the fact that the decrease of steady-state fluorescence intensity was greater than fluorescence lifetime. The fluorescence quenching was profound under longer excitation and emission wavelength. The binding constant (K, L/mol) for Ca2+ to CDOM from terrestrial source ranged from 4.29 to 5.09 (lgK), which was approximately one order of magnitude higher than that of Mg2+ to CDOM (3.86 to 4.56). Fluorescence decay became faster in the presence of Ca2+ and Mg2+. Lifetime distribution of CDOM excited states shifted to small value side in the presence of metal ions, particularly for Ca2+, indicating fluorescence quenching of CDOM mainly through the interaction of Ca2+/Mg2+ with relatively long-lived fluorophores.