A simple and highly sensitive voltammetric method was developed to determine the Shikonin (SHI) content in synthetic and real samples. The method is based on a carbon pate electrode (CPE) modified by the addition of sepiolite clay, TiO2 nanoparticles and multi-walled carbon nanotubes (MWCNTs). Differential puls adsorptive stripping voltammetry (AdsDPV) technique was employed with the electrode as the anode. Furthermore, electrochemical redox properties of SHI were investigated by using cyclic voltammetry (CV) on the same electrode. The CV studies show that SHI has two quasi-reversible oxidation signals at about 0.100 V (P-1) and +0.700 V (P-2) in acidic media. To the best of our knowledge, the oxidation peak P-1 has not been observed in previous voltammetric studies on SHI. We suggest mechanisms that lead to both P-1 and P-2. The peak P-1 was selected for the quantitative analysis of SHI on the electrode concerned, because it is more intense than P-2 (higher peak current, higher sensitivity). P-1 is also the choice in terms of interference effects of several organic compounds likely to be present in biological samples. The anodic peak current of SHI in AdsDPV varies linearly with the concentration range of 0.44-1000 n mol L-1. Dedection (LOD) and quantification (LOQ) limits were calculated as 0.13 n mol L-1 and 0.44 n mol L-1, respectively. The voltammetric technique was applied to SHI analysis in human urine and serum samples and found to produce expectable quality results. (C) 2016 Elsevier B.V. All rights reserved.