We present a highly efficient multichannel microfluidic electrochemical sensor integrated with an electroactive nanocarbon microelectrode for sensitive and selective detection of multiple biomarkers in different biological samples. Our results have shown that ionic liquid-assisted wet spinning followed by tailored growth of metal-organic frameworks and pyrolysis treatment led to structural and molecular engineering of mechanically robust all-carbon microfibers for excellent electrochemical activities. The flexible bottlebrush-like nanocarbon microelectrode features a "stem" of freestanding N, B-codoped graphene fiber and high-density "bristles" of Co, N-codoped carbon nanotube arrays, leading to promoted electrocatalytic mechanism that has been substantiated by density functional theory calculations. The structural characteristics, high catalytic activities, and favorable biocompatibility of the bottlebrush nanocarbon electrodes provide opportunities for multichannel, microfluidic detection of redox-active biomolecules, including hydrogen sulfide (H2S), dopamine (DA), uric acid (UA), and ascorbic acid (AA), and have been applied to on-chip monitoring of H2S and DA released from live cancer cells or neuroblastoma cells and DA, UA, and AA in trace amounts of body fluids such as sweat, finger blood, tears, saliva, and urine, which is of great significance for clinical diagnosis and prognosis in point-of-care testing.