Abstract The COVID-19 pandemic has highlighted the significance of swift and efficient large-scale screening to prevent epidemic outbreaks. Solution-gated graphene transistors (SGGTs) have emerged as a valuable asset in creating point-of-care (POC) sensing platforms for detecting SARS-CoV-2, owing to their notable stability and sensitivity in solution environments. However, the poor interface engineering of sensing electrodes is of great concern for reliable functionalization, leading to poor sensitivity. In this study, we present an interface engineering strategy for realizing the functionalization of SGGT sensing electrodes with high stability and sensitivity to construct a SARS-CoV-2 nucleic acid biosensor. The developed biosensor exhibits the capability to detect ORF1ab gene fragments at a concentration as low as 10−16 M without requiring additional nucleic acid amplification. Furthermore, the entire detection process can be accomplished using a portable biosensing microsystem within 30 minutes. This research holds promise for advancing the study of FET-based biosensors and facilitating their practical application in clinical settings.