5-Hydroxytryptamine (5-HT) is an important neurotransmitter, and its content in the human body is of great significance to human health. In this study, an L-cysteine-terminated triangular silver nanoplate loaded on a MXene (twodimensional transition metal carbide or nitride) (Tri-AgNP/L-Cys/MXene) electrochemical sensor was used to detect 5-HT. As an electrically active amino acid with a sulfhydryl group, L-cysteine (L-Cys) forms a more stable Ag−S bond with silver nanoparticles, which can selectively substitute trisodium citrate (TSC) in TSCcapped triangular silver nanoplates (Tri-Ag-NP/TSC). Due to the good conductivity, biocompatibility, and large surface area, MXenes provide a good platform for loading Tri-AgNP/L-Cys. Under optimized conditions, the concentration range for detecting 5-HT with the sensor is 0.5−150 μM, and the limit of detection (LOD) is 0.08 μM (S/N = 3). For detecting 5-HT in actual serum samples, the sensor also showed a good recovery rate (95.38−102.3%), and the relative standard deviation was 2.2−3.4%.