A new solvent extraction system using a hydrazone derivative such as 1,2-cyclohexanedione bis-benzoylhydrazone (1,2-CHBBH) has been studied to separate and quantify the different fractions of copper present in natural waters. The influence of different variables on the extraction has been studied, such as the type of organic solvent, the reagent concentrations in the extraction and back-extraction solutions, the sample pH, and the extraction time. Under selected conditions, the influence of both organic and inorganic ligands present in natural waters on the efficiency of the extraction process has been also investigated. Thus, the effect of the major anion species present in this kind of sample (Cl-, HCO3-, SO42-, Br-) as well as the effect of humic substances have been analyzed. Differences in the extraction efficiency were observed when organic ligands were presented in the samples. These differences allow the selective separation and quantification of the non-labile copper fraction in natural waters.