In this work, poly(diallyldimethylammonium chloride) (PDDA)-functionalized graphene (PDDA-Gr) was prepared via in situ reduction of exfoliated graphite oxides in the presence of PDDA. The obtained nano-structure was used as a novel immobilization platform for the electrochemical sensor of rutin. PDDA-Gr has superior electrocatalytic activity for the redox of rutin due to its large surface, fast electron transfer character and strong adsorption effect on rutin. The electron transfer rate constant (k(s)) and the electro-active surface area were calculated as 1.59 s(-1) and 0.3324 cm(2) respectively. The saturating absorption capacity (Gamma) value of rutin on modified electrode was calculated to be 2.23 x 10(-9) mol cm(-2). Applying differential pulse voltammetry (DPV), a good linear relationship of the reduction peak current and the concentrations of rutin in the range of 0.0004-1.0 mu M was achieved. The detection limit was 0.04 nM (SIN of 3). Under the optimum conditions, the metabolite of rutin, quercetin can be effectively separated from rutin, which suggested the great potential of the proposed method for the practical application in pharmacokinetic and clinical analyses. The method was successfully applied to the fast determination of rutin in medicine tablets and human plasma with satisfactory recoveries. (C) 2014 Elsevier B.V. All rights reserved.