The exocytotic release of dopamine is one of the most characteristic but also one of the least appreciated processes in dopaminergic neurotransmission. Fluorescence imaging has yielded rich information about the properties of synaptic vesicles and the release of neurotransmitters in excitatory and inhibitory neurons. In contrast, imaging-based studies for in-depth understanding of synaptic vesicle behavior in dopamine neurons are lagging largely because of a lack of suitable preparations. Midbrain culture has been one of the most valuable preparations for the subcellular investigation of dopaminergic transmission; however, the paucity and fragility of cultured dopaminergic neurons limits their use for live cell imaging. Recent developments in stem cell technology have led to the successful production of dopamine neurons from embryonic or induced pluripotent stem cells. Although the dopaminergic identity of these stem cell-derived neurons has been characterized in different ways, vesicle-mediated dopamine release from their axonal terminals has been barely assessed. We report a more efficient procedure to reliably generate dopamine neurons from embryonic stem cells, and it yields more dopamine neurons with more dopaminergic axon projections than midbrain culture does. Using a collection of functional measurements, we show that stem cell-derived dopamine neurons are indistinguishable from those in midbrain culture. Taking advantage of this new preparation, we simultaneously tracked the turnover of hundreds of synaptic vesicles individually using pH-sensitive quantum dots. By doing so, we revealed distinct fusion kinetics of the dopamine-secreting vesicles, which is consistent within both preparations.