In this Article, a rotating droplet system is used for simultaneous detection of dopamine and serotonin. Carbon nanoparticles functionalized with sulfonic groups on the electrode surface enables potential discrimination between the neurotransmitters and the most common interferences, whereas the efficient and low-volume hydrodynamic system helps to lower the detection limit toward physiologically relevant concentrations. Here, we present results with a 10 nM limit of detection for serotonin and a 100 nM to 2 μM linear response range from the system in a sample containing an equimolar concentrations of dopamine and serotonin and 0.5 mM concentration of both uric and ascorbic acids. Demonstrating the practical applicability of this method, we measure the concentration of serotonin in 70 μL of mice blood serum samples without additional pretreatment.