Platelet aggregation in the bloodstream is tightly associated with the secretory function of platelets based on several types of cytoplasmic secretory granules, each sequestering distinct chemical messenger species. Dense-body granules are one prominent type of secretory granule responsible for storing small molecule chemical messengers. Upon platelet activation, the timely and rapid release of these small molecules is critical in facilitating platelet aggregation. Therefore, techniques capable of measuring real-time granule content release are needed to understand the fundamental properties of platelet secretion and aggregation. Existing techniques lack adequate time resolution or require potentially toxic exogenous reagents for real-time measurement of granule content release. Herein, we demonstrate a label-free electrochemical method based on the endogenous electroactive chemical messenger serotonin (5-hydroxytryptamine or 5-HT) for the real-time measurement of dense-body granule secretion from platelet suspensions; fast-scan cyclic voltammetry (FSCV) using carbon-fiber microelectrodes was chosen on the basis of its excellent temporal resolution, high sensitivity, and the ability to provide the electrochemical signature cyclic voltammograms for molecular identification. Real-time serotonin release from thrombin-stimulated human platelet suspensions was successfully measured, and the amount and time course of the bulk serotonin release were found to agree well with data obtained from single platelet measurements, thus confirming accurate characterization of granular secretion. Furthermore, this electrochemical method was applied to study the stimulation-secretion coupling in platelets, serotonin storage and release dynamics with applied pharmacological agents, and chemical messenger storage deficiency in Hermansky-Pudlak Syndrome (HPS) platelets, and the potential of this method to reveal secretion behavior in both normal and diseased platelets has clearly been demonstrated.