A bi-parametric sequential injection method for the determination of copper(II) and zinc(II) when present together in aqueous samples was developed. This was achieved by using a non-specific colorimetric reagent (4-(2-pyridylazo)resorcinol, PAR) together with two ion-exchange polymeric materials to discriminate between the two metal ions. A polymer inclusion membrane (PIM) and a chelating resin (Chelex 100) were the chosen materials to retain zinc(II) and copper(II), respectively. The influence of the flow system parameters, such as composition of the reagent solutions, flow rates and standard/sample volume, on the method sensitivity were studied. The interference of several common metal ions was assessed, and no significant interferences were observed (<10% signal deviation). The limits of detection were 3.1 and 5.6 µg L−1 for copper(II) and zinc(II), respectively; the dynamic working range was from 10 to 40 µg L−1 for both analytes. The newly developed sequential injection analysis (SIA) system was applied to natural waters and soil leachates, and the results were in agreement with those obtained with the reference procedure.