In vivo tracking or in vitro real sample analysis by electrochemistry is one of the most straight and useful methods in biosensor field. However, surface biofouling of electrodes by non-specific protein adsorption is inevitable and usually leads to a decrease in sensitivity. Here, we developed a Nafion-coated porous boron-doped diamond (NAF/pBDD) electrode with hydrophobic nanostructures to minimize the biofouling effect and selectively detect dopamine (DA). Larger active area was obtained by this procedure compared to a bare diamond electrode. The as-prepared electrode shows excellent antifouling property and enrichment capacity toward selective detection of dopamine (DA). The low background current of the BDD electrode and the enhanced signals enables a lower detection limit, 42 nmol L-1, and a wider linear range, 0.1-110 mu mol L-1, for determination of DA in human serum. Additionally, the facile modified electrode demonstrated renewable property and long-term stability due to the fact that the antifouling nanostructures belong to its own.