A versatile reusable electrochemical biosensor has been formulated by using colloidal gold nanoparticle (AuNPs) suspension and conducting polymer poly-(3-amino-5-hydroxypyrazole (poly-AHP) modified carbon paste electrode (AuNPs/poly-AHP/CPE) for detection of an important anti-viral drug valacyclovir (VAL). A thin film of poly-AHP incorporated with homogeneous distribution of AuNPs on the surface of carbon paste electrode is fabricated stepwise by applying one by one. An electrochemical potentiodynamic polymerization of AHP was followed by a potentiostatic electrodissolution–induced deposition of AuNPs on poly-AHP. The sensor surface has been characterized by scanning electron microscopy (SEM), energy dispersive X-Ray spectroscopy (EDX), X-ray powder diffraction (XRD) and UV–visible spectroscopy for surface topological/morphological characteristics. Electrochemical efficiency of the present sensor system towards the detection of VAL is explored by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), differential pulse voltammetry (DPV) and hydrodynamic steady-state current–time analysis, and the fabricated AuNPs/poly-AHP/CPE is recognized as a capable sensor system for electrochemical determination of VAL. Applying DPV under optimized conditions, the anodic peak current exhibited a linear relationship against VAL over the range of 5–80 nM with a detection limit of as low as 1.9 nM and an analysis time of 15 s. Under hydrodynamic conditions mimicking flow-cell analysis, the low detection limit was as low as 2.5 nM (S/N > 4) with rapid analysis time of mere 5 s. Selective determination of VAL was performed in the presence of dopamine and serotonin. Practical utility of the AuNPs/poly-AHP film sensor has been demonstrated for the detection of VAL directly from artificial urine, pharmaceutical formulations and diluted human serum with good recovery limits. © 2021 Elsevier B.V.