The selective separation of molybdenite from copper sulfide concentrate in flotation process is realized using sodium hydrosulfide (NaHS) to depress the chalcopyrite and permit only the flotation of the molybdenite. However, this reagent is a highly toxic and flammable gas. The objective of this research was to study the feasible application of commercial lignosulfonates (LSs) in the separation by froth flotation process of molybdenite and chalcopyrite in seawater to present a novel application for LSs, as well as an alternative reagent to sodium hydrosulfide (NaHS). To achieve this, microflotation, absorbance tests and zeta potential measures were performed at pH 8 in seawater and 0.01 M NaCl. The results obtained in this study showed that it is possible to achieve selective separation of copper and molybdenum in both aqueous media due to high depressant effect of molybdenite and low depression of chalcopyrite in microflotation tests at 10 ppm of LSs, when the collector, PAX, is added prior to the addition of LSs. Absorbance study and zeta potential measurements showed that LSs adhere more to the molybdenite surface in seawater than in freshwater. This is related to the high ionic charge of the media that influences a greater interaction of LSs with the mineral surface.