In this study, we report a portable kit consisting of a portable workstation, gold screen-printed electrode (SPE), 0.45 μm filter membrane, phosphate buffer solution (PBS), and acetic acid (1%) for point-of-use (POU) analysis of nicotine in tobacco. The activated-screen-printed electrode (A-SPE) displayed superior electron transmission efficiency, and the A-SPE without modification was employed for high-performance analysis of nicotine in actual tobacco after simple sample pretreatment. Remarkably, the fabricated nicotine sensor exhibited a broad working range of 10-100 μg g-1, a low limit of detection (LOD) of 6.4 μg mL-1, good stability, selectivity, and practicality under the optimal conditions. The method was applied to the determination of nicotine in (spiked) samples. Satisfactory recovery results demonstrated that the as-prepared portable kit method with outstanding electrocatalysis ability was feasible for analysis of nicotine in tobacco. Moreover, the values obtained using the A-SPE were in good agreement with those determined by gas chromatography-flame ionization detection (GC-FID), which confirms the feasibility and validity of the present method. The results of the as-proposed portable kit provided a new strategy for analyzing nicotine in actual tobacco samples.