An electrochemical separation procedure has been developed for the speciation of Cr(VI) and V(V) in water samples. The sensing technique was based on the direct linear sweep voltammetric reduction of Cr(VI) and V(V) on a multi-walled carbon nanotube-neutral red-gold nanoparticles (MWCNTs-NR-AuNPs) modified commercially available screen-printed carbon electrode (SPCE). In this study, MWCNTs attached on the surfaces of NR-AuNPs via the covalent bonding between the carboxylic acid of MWCNTs and the free amine group of neutral red. The peak to peak separation of V(V) and Cr(VI) was about 269 mV, Total chromium and vanadium were determined after oxidation of Cr (III) to Cr(VI) and V(IV) to V(V) by potassium permanganate oxidation. The linear range of Cr(VI) and V(V) was 0.4-80 mu M and 3-200 mu M with the detection limits of 0.025 mu M and 0.42 mu M (S/N = 3), respectively. The recoveries of Cr(VI), Cr(III), V(V) and V(IV) were 97-99%, 96-98%, 97-99% and 95-98%, respectively, HorRat were calculated to 0.60-0.91 for Cr(VI), 0.55-1.11 for Cr(III), 50-1.16 for V(V) and 0.63-1.23 for V(IV), respectively. The proposed electrochemical separation procedure shows appropriate sensitivity and offers an alternative to the simultaneous detection and speciation of target metal ions.