A novel, simple, cost-effective, easy-to-prepare, and timesaving electrochemical sensor based on gold clusters decorated yellow 2G polymer composite film was fabricated and comprehensively characterized for the determination of dopamine and nicotine in real samples. When gold clusters were modified on the yellow 2G polymer film, a signal increase of 204% for dopamine and 526% for nicotine was obtained compared to the activated bare pencil graphite electrode. The signals at 90mV for dopamine and 780mV for nicotine were utilized as analytical responses. The fabricated platform serves noteworthy detection sensitivity values of 79.02μA.μM−1.cm−2 and 14.61μA.μM−1.cm−2 with a notable limit of detection values of 58.8nM and 7.2nM for dopamine and nicotine, respectively. The electrochemical method has linear ranges of 0.075–5µM and 6–10µM for dopamine alone, 0.01–10µM and 12–25µM for nicotine alone, 0.01–10µM and 12–50µM for nicotine in the presence of dopamine, 0.2–10µM and 12–60µM for dopamine in the presence of nicotine, and 0.4–2, 2–10 and 12–90µM for dopamine and 0.2–8, 10–20 and 25–90µM for nicotine at the increasing amounts of each substance. Moving forward, the developed method exhibits splendid accuracy and selectivity for determining dopamine and nicotine in biological samples of human saliva, serum, and urine.