Metal speciation studies are of great importance in assessing metal bioavailability in aquatic environments. Functionalized membranes are a simple tool to perform metal chemical speciation. In this study, we have prepared and tested a polymer inclusion membrane (PIM) made of the polymer cellulose triacetate (CTA), the extractant di-(2-ethylhexyl) phosphoric acid (D2EHPA), and the plasticizer 2-nitrophenyloctyl ether (NPOE) as a sensor for Zn and Cu complexation studies. This PIM, incorporated in a device with an 0.01 M HNO3 receiving solution, is shown to effectively transport free metal ions, and it is demonstrated that the presence of ligands that form stable complexes with divalent metallic ions, such as ethylenediaminetetraacetic acid (EDTA) and humic acid (HA), greatly influences the accumulation of the metals in the receiving phase due to the increasing metal fraction complexed in the feed phase. Moreover, the effect of major ions found in natural waters has been investigated, and it is found that the presence of calcium did not decrease the accumulation of either Zn or Cu. Finally, the PIM sensor has been used successfully to evaluate metal complexation in a river water affected by Zn pollution.