Dopamine (DA) is a critical neurotransmitter whose dysregulation is closely associated with Parkinson's disease; however, its reliable detection in blood remains challenging because of its extremely low concentration and severe matrix interference. Herein, we report a highly sensitive electrochemical sensor based on a hybrid nanocomposite consisting of carbon nitride quantum dot-supported Ru nanoparticle-decorated carbon nanotubes (Ru-CNQD/CNT) integrated with hollow carbon spheres (HCS) for DA detection. Owing to the synergistic combination of the high activity of Ru-CNQD/CNT and the favorable enrichment capability of HCS, the proposed sensor exhibits excellent analytical performance, featuring a wide linear response range from 0.001 to 50.0 μM and a low detection limit of 0.13 nM. The electrochemical platform was further applied to monitor DA release from PC12 cells under high-potassium stimulation, enabling dynamic electrochemical tracking of neurotransmitter secretion. Importantly, quantitative analysis of DA in human serum samples demonstrates that the sensor can effectively differentiate Parkinson's disease patients from healthy individuals. Overall, this work establishes an efficient and ultrasensitive electrochemical strategy for DA detection in complex biological matrices, showing strong potential for neurotransmitter monitoring, DA-related disease assessment, and fundamental neurochemical research.