A conductive diamond-like carbon (DLC) electrode was prepared by coating a conductive DLC thin film onto a silicon substrate surface using high-power impulse magnetron sputtering (HiPIMS) and ionization deposition methods. The resulting DLC electrode showed a wide potential window of ~3 V. Its background current was slightly larger than that of the boron-doped diamond (BDD) electrode and much smaller than that of the glassy carbon electrode, indicating that the DLC electrode has electrochemical properties similar to those of the BDD electrode. Electrochemical measurements in phosphate buffer solution (PBS) containing bovine serum albumin (BSA) did not result in electrode fouling, suggesting that the DLC electrode is suitable for highly sensitive electrochemical measurements in biological fluids. Furthermore, conductive DLC was deposited on the tip of a glass needle to fabricate a DLC microneedle (DLC-MN) electrode with a small tip diameter of 600 nm. The DLC-MN electrode exhibited typical microelectrode behavior and was used to determine ascorbic acid and theophylline in PBS containing BSA. With its small tip diameter and excellent fouling resistance, the DLC-MN electrode holds great promise for facilitating highly sensitive and stable local electrochemical sensing or monitoring in vivo.