In order to research the effect of water quality on the separation of specularite and chlorite, the effect and mechanism of Ca2+ and Mg2+ on the flotation behavior of specularite and chlorite using dodecylamine (DDA) as the collector were studied via micro-flotation tests, Zeta potential measurements, Boltzmann theoretical analyses, solution chemistry calculation and molecular dynamics simulation in deionized water and tap water flotation environment, respectively. The results of micro-flotation tests demonstrated that specularite and chlorite were depressed to some extent in tap water. Compared with the deionized water system, the recovery rates of specularite and chlorite decreased by 8.01 and 8.99 percent point, respectively. In simulated tap water environment, Ca2+and Mg2+ made the recovery rate of specularite decreased by 11.91 and 18.88 percent point, respectively, while decreased by 7.44 and 15.45 percent point, respectively, for chlorite. The inhibitory effect of tap water on specularite and chlorite flotation mainly caused by the existence of Ca2+ and Mg2+. The depression effect of Ca2+ and Mg2+ on specularite was stronger than that of chlorite, and the depression effect of Mg2+was more obvious than Ca2+. The results of mechanism detection indicated that the adsorption of Ca2+ and Mg2+ shifted the surface potential of the two minerals to higher values, wakened the electrostatic adsorption reaction between DDA and the two minerals, decreased the contact angle, hydrophobicity and the concentration of RNH3+ in interface layer, increased the adsorption distance between DDA and the two minerals, increased the loose degree of DDA, which in all depressed the flotation of specularite and chlorite. The research contents provide theoretical basis of disturbance law and elimination methods of water quality for iron ore flotation.