A highly sensitive and selective electrochemical sensor of dopamine (DA) has been developed by employing carboxylated carbonaceous spheres to modify glassy carbon electrodes (GCEs). Scanning electron microscopy (SEM) and Fourier transform infrared (FT-IR) spectroscopy were used to characterize as-prepared carbonaceous spheres. The results show that the diameter of carboxylated carbonaceous spheres is uniformly 500 nm and that their surfaces mainly expose carboxyl groups with negative charges. Electrochemical measurements demonstrate that carboxylated carbonaceous spheres greatly improve the accumulation of positively charged dopamine, leading to good sensing performance on a modified GCE. Through applying the differential pulse voltammetric approach, linear calibration curves were obtained in a range of about 0.1 to 40 mu M with a detection limit down to 30 nM. Furthermore, depending on the charge-based discrimination, the modified electrode displays good selective detection of DA and reliable anti-interference to UA and glucose besides a weak and negligible response to AA. Therefore, the carboxylated carbonaceous sphere introduced here is a good candidate to develop electrochemical sensors for the sensitive and selective detection of DA.