Cotinine (CO) is the principal metabolite of nicotine, and it is used as a biomarker to monitor the exposure of an individual to tobacco. This paper sought to present the use of a boron-doped diamond (BDD) electrode for the electrochemical characterization of CO by voltammetric techniques and its quantification in saliva samples by multiple-pulse amperometry (MPA) in a flow injection analysis (FIA) system. In phosphate buffer solution 0.1 mol L-1 (pH 7), the CO presented two electrochemical processes, one anodic at +1.8 V and one cathodic at -1.2 V (vs. Ag/AgCl). The second process was dependent on the first and both processes were irreversible. It was found that CO electrochemical processes are controlled by diffusion and that two-protons and two-electrons are involved in the oxidation reaction. In determination of CO in saliva samples, the MPA detection was used, applying four potential pulses: (1) at +1.8 V/400 ms, generator potential pulse for oxidation of CO; (2) at -1.0 V/500 ms, potential pulse for remove possible interferents; (3) at -1.2 V/30 ms, collector potential pulse for reduction of the product generated and CO quantification; (4) 1.45 V/300 ms, potential pulse for cleaning BDD electrode. The proposed method attained an analytical frequency of 36 determinations per hour with a relative standard deviation of 1.47% for 10 measurements of 1.0 x 10(-5) mol L-1 CO. A good linear range from 0.5 to 100 mu mol L-1 was obtained with a detection limit for CO estimated at 0.06 mu mol L-1. Approximately 100% of the CO added to saliva samples was recovered. Therefore, this work presents, for the first time, the electrochemical behavior of the CO at an electrode as well as a simple, fast and low-cost method for monitoring the degree of exposure to tobacco by CO determination in saliva samples using the FIA-MPA technique. (C) 2016 Elsevier Ltd. All rights reserved.