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In this work, gold nanoparticles were biosynthesized via Plectranthus amboinicus leaf extract as the reducing agent. A series of techniques were used for sample analysis. The biosynthesized gold nanoparticles (bAuNPs) are a uniform size with a spherical shape. The FTIR analysis reveals the presence of many oxygen-containing functional groups on the bAuNP surface. The cyclic voltammetry and electrochemical impedance spectroscopic characterizations reveal that while the bAuNPs have a slightly lower conductivity than chemically synthesized AuNPs (cAuNPs). However, the bAuNPs have a superior electrocatalytic performance toward nicotine reduction. After optimization, the bAuNP-modified SPE could detect nicotine linearly from 10 to 2,000 μM with a low detection limit of 2.33 μM. In addition, the bAuNPs/SPE have been successfully used for nicotine-containing-product analysis.