Graphene field-effect transistor (G-FET) biosensors require precise surface functionalization to achieve high sensitivity and stability. However, existing methods lack spatial control capabilities and may cause extensive damage to graphene’s electrical properties. In this study, we introduce an atomic force microscopy-based precision functionalization technique, enabling site-specific modification of graphene via redox reactions at a biased probe tip. This approach allows stable, localized immobilization of cTnI-specific aptamers, minimizing graphene damage and yielding high-performance G-FET biosensors. The resulting sensor detects cardiac troponin I at concentrations as low as 0.01 pg ml, exhibiting linear resistance response and strong selectivity in concentration gradient assays. This technology provides a scalable solution for constructing multiplexed biosensing within a single sensor.