Development of electrochemical sensors for important drugs such nicotine (an addictive drug) is important for the society. This study reports the electrochemical detection of nicotine at a carbon nanofiber/poly (amidoamine) dendrimer modified glassy carbon electrode. The carbon nanofiber (CNF) modified GCE was prepared by drop-coating followed by the electrodeposition of generation 4 poly (amidoamine) succinamic acid dendrimer (PAMAM) to form the sensor - CNF-PAMAM GCE. Characterization of prepared materials and modified electrodes was carried out using Fourier transmission infrared spectroscopy, field emission scanning electron microscopy, transmission electron microscopy, Raman spectroscopy, cyclic voltammetry, electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV). The CNF-PAMAM composite was confirmed by microscopy. A marked reduction in charge transfer resistance and increase in current of the CNF-PAMAM GCE in comparison to the bare electrode showed a synergic improvement electrochemical response because of the CNF-PAMAM nanocomposite. The CNF-PAMAM demonstrated an enhanced performance in the oxidation of nicotine in comparison to the bare GCE by shifting the anodic potential Epa of nicotine from 0.9 V to 0.8 V. The electrochemical sensor achieved a detection limit (LOD) of 0.02637 μM in the concentration range of 0.4815-15.41 μM of nicotine in 0.1 M PBS at pH 7.5. The sensor ability to determine nicotine in real samples was assessed in cigarettes obtaining recovery percentages of 88.00 and 97.42%. The sensor demonstrated selectivity toward nicotine in the presence of interferences. Finally, the method was validated by ultraviolet-visible spectroscopy analysis.