A thin and conductive polytyramine (Pty) film was deposited on the surface of a glassy carbon electrode (GCE) in phosphate buffer solution (pH = 4.07) by reversible potentiodynamic scans in the range of -200 mV to 1000 mV. The reactivity of the polymeric film was characterized using electrochemical methods in the presence of redox couple species and scan rate variations. The amount deposited during the entire repetitive cycles approach 12.96 ug. The mean surface roughness estimated by Scanning Tunneling Spectroscopy (STM) technique is 0.7 nm interestingly insignificant to indicate the formation of a very thin layer. The modified electrode exhibited a substantial reactivity and high sensitivity in the oxidation of dopamine (DA) and ascorbic acid (AA). The symmetry and peak separation for above biological components were identified and the expected interference capacity by either component has been advantageously eliminated to a larger extent. The present working system provides a simple and fundamental approach for the simultaneous and selective determination of DA and AA with a detection limit brought down to 214 nM and 414 nM, respectively but the catalytic activity and separation capacity of the Pty/GC modified electrodes towards the simultaneous determination of DA, AA and uric acid (UA) were not feasible due to interference effect. The presence of UA in the electrolytic mixture with AA and DA inhibits the electron transfer kinetics of DA species which diminishes its peak current. To improve the catalytic activity of the modified electrode, tris(2,22-bipyridyl) ruthenium(II) (Ru(bpy)3]2+ was incorporated on Pty film at GCE. The results show that Ru*2/Pty/GCE is able to successfully detect DA in the presence of large concentrations of AA and UA without any interference. Also, electrode fouling due to the accumulation of oxidation products was not identified as the electrode system sustain good activity and stability even at high initial concentrations of the species.