The rapid spread of infectious diseases, exemplified by the well-known case of COVID-19, which has led to a global pandemic, has garnered increasing attention toward the development of simple and efficient biosensors for diagnosis. In this study, a field-effect transistor (FET)-based biosensor offers an alternative and promising solution for the diagnosis of clinical COVID-19. Molybdenum disulfide (MoS2) in a channel FET modified with a probe complementary DNA (cDNA) can sensitively detect selected specific sequences from SARS-CoV-2 through hybridization. MoS2 was modified with probe cDNA and exhibited a linear concentration range of 1 fM to 0.1 nM in both phosphate-buffered saline (PBS) and serum samples, with low limits of detection (LOD) of 0.21 and 0.73 fM, respectively. In addition, it demonstrated a rapid response time of 3 min in clinical samples. The developed biosensor is capable of analyzing RNA from 19 clinical samples. These results indicate that the biosensor successfully distinguished between healthy and infected samples, which is consistent with the RT-PCR results (kappa coefficient = 1). Additionally, a notable difference was observed between the RdRp samples of SARS-CoV-2 and SARS-CoV. Therefore, we believe that this study offers a reliable and appealing option for the diagnosis of COVID-19.