An electrochemical assay for sensing NO in biological systems is described in this paper. The ferrocene mediated reduction of NO, facilitated by the gold nanocomposite modified glassy carbon electrode is followed by an amperometric procedure. The analytical protocol involves the modification of a glassy carbon electrode by an overlayer of Au nanocomposites prepared through galvanic reduction. Additional overlayers can be built on the surface by repetition of the procedure. The modification leads to the decrease of the over-potential required for the analysis and results in a non-biofouling surface. Since the procedure is based on the electrochemical reduction of NO, the potential interferences from species like dopamine, ascorbic acid, etc., are overcome. The sensitivity, detection limit and response time achieved through this protocol for the modified electrode containing three Au overlayers are 0.03 nA/nM, 25.75 nM and <5 s. Analysis of NO has been carried out in real samples like liver extract, peripheral blood mononuclear cells (PBMCs) and miconazole nitrate ointment and the values obtained are comparable with that obtained by Griess analysis.