The key problem that limits the practical applications of nonenzymatic electrochemical sensors in biological media, is the biofouling and chemical fouling of electrodes due to the adsorption of biological molecules and oxidation (reduction) products. Electrode fouling will cause low accuracy, poor stability, and low sensitivity. Here, a simple and efficient antifouling electrode was demonstrated for electrochemical sensing based on covalent-organic framework (COF) TpPA-1 and carboxylic multi-walled carbon nanotubes (CNT) composites. COF TpPA-1 possesses abundant hydrophilic groups, which assisted the dispersion of CNT in water and formed uniform composites by π-π interaction. In addition, the introduction of CNT into the composites improved the electron transfer rate of COF TpPA-1. The antifouling interface was characterized by electrochemistry, contact angle measurement, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and X-ray photoelectron spectroscopy (XPS). The electrode showed good chemical and bio-fouling resistant performance for the electrochemical detection of β-nicotinamide adenine dinucleotide (NADH) and uric acid (UA) in real serum samples.