In situ mercury film coated graphite pencil electrode (MF/GPE) was prepared and applied for selective and sensitive electrochemical determination of anticancer drug lomustine (LMT). Parameter optimization for the preparation of MF/GPE was examined. The bare and modified GPEs were characterized by scanning electron microscopy (SEM), cyclic voltammetry and square wave voltammetry. With pH 5.0, Britton-Robinson (BR) buffer and in the presence of 0.5 M sulfate ions as indifferent supporting electrolyte, LMT yields a well defined and sensitive reduction peak at the MF/GPE. The electrochemical parameters such as the charge value (Q), surface concentration (Gamma), electron transfer coefficient (alpha) and the standard rate constant (k(s)) for the reduction of LMT at the MF/GPE were calculated. The achieved limits of detection and quantification were 8.13 x 10(-8) M and 2.71 x 10(-7) M by square wave cathodic adsorptive stripping voltammetry (SWCASV), respectively. The modified MF/GPE was used as a sensor for the detection of LMT in human blood and urine samples with good accuracy and precision. (C) 2015 Elsevier B.V. All rights reserved.