Passivation of electrode surface and tedious reconstruction of biosensing architectures have long plagued researchers for the development of electrochemical biosensors. Here, we report a novel self-cleaning electrode by modifying the commonly used working electrode with superhydrophobic and conductive nanocomposite. Owing to the superhydrophobicity and the chemical stability, the electrode avoids passivation result from both adsorption of molecules and oxidation in air. The high conductivity and the high effective area also allow the achievement of enhanced electrochemical signals. On the basis of comprehensive studies on this novel electrode, we have applied it in the fabrication of refreshable electrochemical biosensors for both electro-active and electro-inactive targets. For both cases, detection of the targets can be well performed, and the self-cleaning electrode can be refreshed by simply washing and applied for successive measurements in a long period.