This work reports an electrochemical sensing framework for nicotine determination based on glassy carbon electrode (GC) immobilized with Fe(bpy)32+ (where bpy is 2,2’‐bipyridyl) supported by Nafion and multi‐walled carbon nanotubes (Nf‐MWCNTs). Fe(bpy)32+ immobilized Nf‐MWCNTs modified GC (GC/Nf‐MWCNTs/Fe(bpy)32+) manifests stable redox peaks, characteristics of Fe(bpy)32+. The GC/Nf‐MWCNTs/Fe(bpy)32+ exhibits effective electrochemical oxidation of nicotine, diminishing the overpotential relative to GC/Nf‐MWCNTs. The limit of detection is 0.1 μM (experimentally observed) with two different linear calibration ranges between 0.1 to 600 μM and 600 to 3000 μM. Electrocatalytic responses observed at GC/Nf‐MWCNTs/Fe(bpy)32+ indicate superior performance for nicotine determination with acceptable selectivity, stability, and reproducibility. Additionally, the nicotine present in real samples such as beedi and tobacco are also analyzed with satisfactory recovery percentages.