Using seawater for mineral flotation conserves fresh water resources and offers significant environmental and economic benefits. However, the adverse effect of seawater restricts its application in mineral flotation. The regulators can eliminate the adverse effect caused by seawater during flotation separation of lead and copper. In this paper, the flotation separation of galena and chalcopyrite in seawater was tested. Compared with those in deionized water, the lead grade of concentrate in seawater flotation declined from 50.10% to 48.90%. Meanwhile, the recovery of galena declined from 74.33% to 60.58% and the SI value declined from 1.565 to 1.324. With the addition of SHMP (sodium hexametaphosphate), SS (sodium silicate) and EDTA (ethylenediamine tetraacetic acid), the SI value increased from 1.324 to 1.923, 2.002 and 1.939, respectively. Noteworthy, when EDTA was excessive, the grade of Pb was 60.80%, the recovery of galena was 60.86% and the SI value was 2.113. X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, Atomic force microscopy, Density functional theory calculation and E-DLVO calculation verified the micro-flotation results and revealed the mechanism of action.