In this work, we explore the performance of combined reference/working electrodes modified with ruthenium oxide and Poly(3,4-ethylenedioxythiophene) (PEDOT) in the detection of nitric oxide with the goal to measure nitric oxide at the level of single or collective cultured cells. The synergistic effect of the electrocatalytic activity of ruthenium oxide and the enhanced surface area for catalytic activity provided by the polymer greatly enhanced the analytical performance of our sensors in terms of sensitivity, selectivity and stability. With the incorporation of a layer by layer method of electrodeposition we attained a normalized sensitivity of 8.38E-4 pA/nM/μM2 and 1.16E-2 pA/nM/μM2 towards NO in the linear range of 0.2-1.6 mM and 2-16 nM respectively, at an applied potential of 0.8 V vs Ag/AgCl and a detection limit in the vicinity of 500 pM. In order to improve the selectivity of our sensors we coated the surface with nafion and also an ionic liquid composite and lowered the applied to 0.6 V vs. Ag/AgCl. We also measured the response to NO using Differential Pulse Amperometry to further decrease response from biological interferents especially nitrite and ascorbic acid.