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Sensing targeted tumor markers with high sensitivity provides vital information for the fast diagnosis and treatment of cancer patients. A vascular endothelial growth factor (VEGF₁₆₅) have recently emerged as a promising biomarker of tumor cells. The electrochemical aptasensor is a promising tool for detecting VEGF₁₆₅ because of its advantages such as a low cost and quantitative analysis. To produce a sensitive and stable sensor electrode, nanocomposites based on polyaniline (PANI) and carbon nanotube (CNT) have potential, as they provide for easy fabrication, simple synthesis, have a large surface area, and are suitable in biological environments. Here, a label-free electrochemical aptasensor based on nanocomposites of CNT and PANI was prepared for detecting VEGF₁₆₅ as a tumor marker. The nanocomposite was assembled with immobilized VEGF₁₆₅ aptamer as a highly sensitive VEGF₁₆₅ sensor. It exhibited stable and wide linear detection ranges from 0.5 pg/mL to 1 μg/mL, with a limit of detection of 0.4 pg/mL because of the complementary effect of PANI/CNT. The fabricated aptasensor also exhibited good stability in biological conditions, selectivity, and reproducibility after several measurement times after the dissociation process. Thus, it could be applied for the non-invasive determination of VEGF, in biological fluid diagnosis kits, or in an aptamer-based biosensor platform in the near future.