Highly sensitive and selective detection of various biomolecules is critical for biological research and diagnostics. Herein, we have fabricated an in situ hierarchically self-assembled hybrid electrode based on graphene nanoplatelets (GNP) functionalized with silver nanorods (AgNRs) followed by electrochemical polymerization of 4-amino-1-1'-azobenzene-3,4'-disulfonic acid dye (acid yellow 9, poly(AY)) for in vitro electroanalysis of biomolecules in biological samples. The UV-vis spectroscopy, FT-IR, XRD and FE-SEM were used to characterize the AgNRs and its incorporation into the graphene nano-interfaces. The GNP/AgNR/poly(AY) nanocomposite (NC) hybrid interface was introduced to modify the surface of glassy carbon electrode (GCE) to study in vitro electroanalysis of important biomolecules such as dopamine (DA) and ascorbic acid (AA) using cyclic voltammetry (CV) and amperometry. The GNP/AgNR/poly(AY) NC coated electrode exhibited high electrocatalytic activity towards DA and AA in physiological condition (pH similar to 7) and separated their oxidation peaks at different potentials with reasonably good peak separation. As-prepared sensor showed linear response for the electro-catalytic oxidation of DA and AA from 1 to 200 mu M with a detection limit of 0.42 mu M and 0.88 mu M, respectively. Furthermore, the effects of pH, scan rate and interferences on the detection of DA using human serum and urine samples are reported. Thus, the GNP/AgNR/poly(AY) hybrid film modified electrode may be utilized for the development of biomolecule transducers of various disease biomarkers. (C) 2017 Elsevier B.V. All rights reserved.