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In this paper, we developed a low operation voltage and a single reaction step of a graphene field-effect transistor (GFET) biosensor device to detect alanine aminotransferase (ALT). The device is fabricated using large-area graphene thin films by means of chemical vapor deposition. The GFET device operated at 0.1V drain voltage and −0.1 to 0.3V sweep gate voltage exhibited a high pH sensitivity of 23.12mV/pH, a low hysteresis voltage of 1.2mV, and a small drift rate of 4.74mV/h. In this research, the hybrid configuration of l-alanine and α-ketoglutarate immobilized on the graphene membrane was proved to be able to detect ALT with good linearity (R²=0.99) in the ALT concentration range of 10–100U/L.