Nanocrystalline diamonds have recently gained great attention to circumvent the current hurdles, with their appealing properties such as high-surface-area to volume ratio, lowbackground current, wide potential window, biocompatibility, and chemical stability. The nanocrystalline diamonds electrolyte-gated field-effect transistor (NCD-EGFET) can operate directly in solution without involving gate oxides in bringing the hydrogen-tethered moieties and facilitates the p-type surface conductivity. This research investigated on Trans-activator of transcription (Tat) protein; a powerful viral gene activator that plays a pivotal role in the primary stage of the human immunodeficiency virus type 1 (HIV-1) replication. Dosedependent interactions of HIV-1 Tat on NCD-EGFET-based RNA aptamer sensing surface were monitored and attained the detection down to 10 fM. The linear regression curve with 3σ estimation professed the sensitivity range to be 31.213 mV/log10 [Tat Concentration]M and the limit of detection of 6.18 fM. The selectivity analysis of NCD-EGFET was conducted with different proteins from HIV (Nef and p24) and Bovine Serum Albumin. Furthermore, to practice in the clinical application, HIV-1 Tat was spiked into the human blood serum and it displayed the genuine non-fouling interaction with the aptamer. The attained highperformance signal enhancement with nanocrystalline diamond-biosensing aids to circumvent the issues in the current diagnosis.