As a growing aspect of materials science, enormous numbers of synthesis routes have been identified to produce nanomaterials, particularly through simple methodologies. For the first time, a flexible self-assembled AuNPs on poly diallyldimethylammonium chloride (PDDA) binded PANI disposable strip sensor is demonstrated in this work. The flexible strip electrodes are prepared by using polyester overhead projector transparent films (OHP), polyaniline (PANI) conducting polymer was deposited on the chemically activated OHP surface. Later, gold nanoparticles (AuNPs) were self-assembled onto the electro-statically binded PDDA-PANI matrix. Owing to the strong electrostatic interaction between the positively charged PDDA molecules and the negatively charged polyaniline play a crucial role in the formation of nanocomposite films. Moreover, the increased the adhesion between the AuNP and the substrate through PDDA anticipated improved stability of the modified electrodes. The structural, morphological and chemical composition of the modified flexible strips were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), Transmission electron microscopy (TEM), X-ray photoelectron microcopy (XPS) and Fourier Transform Infra-red Spectroscopy (FT-IR) techniques . Cyclic voltammetric, alternating-current impedance, and amperometric methods were used to investigate the catalytic properties of the assembled sensor strips. The newly fabricated AuNPs/ PDDA/PANI/OHP hierarchical nanostructures are produce favorable microenvironment for electron transfer. The higher electrocatalytic performance characteristics were observed for the AuNPs/ PDDA/PANI/OHP modified electrodes, which are attributed to the synergistic effects of the conducting polymer acting as an electron facilitator and the nanostructured Au film act as catalytic surface. These disposable, flexible and low-cost sensors strips offered alternative for electrode materials for electrochemical biosensor.