, an essential neurotransmitter, regulates numerous physiological processes in the human body. Imbalances in 5-HT levels contribute to various health conditions, including neurodegenerative diseases, depression, diabetes mellitus, and serotonin syndrome. 5-HT also functions as a growth factor for tumor cells, influencing various stages of tumor development.Measuring 5-HT concentrations in biological fluids such as blood and serum could facilitate early diagnosis of these conditions. Traditional label-free colorimetric aptasensors often struggle to detect small molecules due to false positives from nonspecific interactions with gold nanoparticles (AuNPs). To address this issue, we propose a novel colorimetric sensing method for specific serotonin detection by eliminating nonspecific AuNP interactions. By incorporating a complementary aptamer hybridization step, our label-free aptamer-based method can selectively detect serotonin while avoiding nonspecific AuNP interactions. We optimized detection conditions and established a calibration curve for 5-HT detection, achieving a limit of detection (LOD) of 2.2 nM. Our method shows high selectivity and sensitivity, even for structurally similar compounds such as dopamine and tryptophan. It successfully quantifies 5-HT directly in complex biological samples with minimal preparation, achieving average recoveries between 92 and 108% (RSD: 9.5−12.9%; n = 5). This label-free aptamer-based approach shows promise for the reliable detection of 5-HT and other small molecules in real biological samples.