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The COVID-19 pandemic has taught us valuable lessons, especially the urgent need for a widespread, rapid and sensitive diagnostic tool. To this, the integration of bidimensional nanomaterials, particularly graphene, into point-of-care biomedical devices is a groundbreaking strategy able to potentially revolutionize the diagnostic landscape. Despite advancements in the fabrication of these biosensors, the relationship between their surface biofunctionalization and sensing performance remains unclear. Here, we demonstrate that the combination of careful sensor fabrication and its precise surface biofunctionalization is crucial for exalting the sensing performances of 2D biosensors. Specifically, we have biofunctionalized Graphene Field-Effect Transistor (GFET) sensors surface through different biochemical reactions to promote either random/heterogeneous or oriented/homogeneous immobilization of the Anti-SARS-CoV-2 spike protein antibody. Each strategy was thoroughly characterized by in-silico simulations, physicochemical and biochemical techniques and electrical characterization. Subsequently, both biosensors were tested in the label-free direct titration of SARS-CoV-2 virus in simulated clinical samples, avoiding sample preprocessing and within short timeframes. Remarkably, the oriented GFET biosensor exhibited significantly enhanced reproducibility and responsiveness, surpassing the detection sensitivity of conventional non-oriented GFET by more than twofold. This breakthrough not only involves direct implications for COVID-19 surveillance and next pandemic preparedness but also clarify an unexplored mechanistic dimension of biosensor research utilizing 2D-nanomaterials., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)