Lyme disease is an infectious disease caused by the Borrelia burgdorferi bacterium. Early diagnosis of Lyme disease could prevent patients from developing serious side effects such as chronic arthritis and permanent neurological disorders. Lyme disease diagnosis is currently held back by a lack of reliable tools that are sufficiently sensitive and specific to allow early stage detection. Here, we demonstrate all electronic nano-biosensors for multiplexed detection of antigens of B. burgdorferi at concentrations as low as 2 pg ml-1. The sensors are based on graphene field-effect transistors (GFETs) coupled with genetically engineered antibody fragments. Single-chain variable fragment (scFv) antibodies are used to obtain a closer proximity of the target-binding event to the graphene sensor surface and for higher immobilization density. When compared to GFET nano-biosensors that use the parental immunoglobulin G (IgG) antibodies, scFv GFET nano-biosensors achieve approximately a 4000 × improvement to the limit of detection. We also demonstrate multiplexed detection of B. burgdorferi antigens through site-specific immobilization of scFvs on GFET arrays, which can potentially reduce the false-positive diagnosis ratio of Lyme disease. This work offers a pathway towards point-of-care detection of Lyme disease at an early stage.