From clinical point of view, it is necessary to develop a reliable detection method of Ledipasvir drug which is a promising medication regimen to treat HCV infection. So, an electrochemical sensor based on NiO nano-flakes (NiO NFs) and activated charcoal (ACH) as natural carbon material was developed for this propose. The characterization of ACH revealed its nanostructure. Its crystallites are composed of 3-8 parallel plane layers of graphite (graphene like structures) with interlayer distance and crystallite size of 0.37 and 0.90 nm, respectively. Highly-crystallized pure NiO NFs with crystallite size of 14 nm and interplanar distance of 0.21 nm were prepared. ACH/CPE afforded 2-fold enhancement in the electrochemical response towards oxidation of ledipasvir exceeds graphene/CPE. ACH's high surface area, porous structure and edge-plane-like defective sites of its graphene platelets result in good adsorptive and electron transfer efficiency towards ledipasvir. Among bare CPE, ACH/CPE, NiO NFs/CPE and [NiO NFs-ACH]/CPE, the electrochemical properties of [NiO NFs-ACH] nanocomposite were superior to the individual ACH and NiO NFs as platforms. [NiO NFs-ACH]/CPE was successfully applied for ultra trace determination of ledipasvir in human plasma with LOD value of 0.55 nM. This sensor is eco-friendly, inexpensive, sensitive and suitable for clinical applications.