Oxygen-containing groups at GO edges or surface greatly influence the electrochemical performance of graphene in terms of the heterogeneous electron transfer rate which can be either advantageous or detrimental towards the sensing of a target analyte 17 and influenced by the percentage of mass incorporations of GO in screen-printed electrodes 18, and by the amount of C/O moieties dominating the voltammetric response 19. For most applications including biosensors, large graphene quantities with controlled amount of defect, edge and basal planes are required. Several methods have been used for preparation of graphene 13, 14 and the graphene oxide based materials 15, 16. Graphene oxide has shown a variety of potential applications in nanoelectronics 3, protective coatings 4, polymer composites 5, catalysis 6, energy storage devices 7, drug delivery, optics 8 as well as sensing and biosensing platforms 9, 10, 11, 12. Graphene and related materials have drawn great research interest in recent years because of their exceptional electrical, mechanical and thermal properties 1, 2. Our results demonstrate that controlling the size of GO sheets may have a profound impact in specific biosensing applications. GO sheet size could enhance or inhibit the sensitivity of the graphene-based electrochemical sensors. However, for the β-lactoglobulin immunosensors, the optimum signals were observed at intermediate GO sheet size. In contrast, for the aptasensor fabricated using covalent immobilization, the binding signal variation decreased with increasing GO sheet size. We found that the aptasensors fabricated using physical adsorption, the binding signal variation was dramatically increased with increasing the GO sheet size. The resulting aptasensors and immunosensors are fabricated by using covalent attachment and physical adsorption. As proof of concept, the sensing performance of these GO samples was probed using a well-known ssDNA aptasensor against microcystin-LR toxin and an immunosensor against β-lactoglobulin. We separated different GO sheets sizes, and we characterized them via atomic force, scanning electron, Raman and X-ray photoelectron spectroscopies and solid state nuclear magnetic resonance (NMR). Here, we investigate the direct effect of GO sheets sizes on biosensor performance. However, the size of the resultant GO sheets changes from the parent graphite yielding a polydispersed solution of sizes ranging from a few nanometers to tens of micrometers. Bulk quantities of graphene can be synthesized by oxidation of graphite to graphite oxide and subsequent exfoliation to graphene oxide (GO). The integration of graphene materials into electrochemical biosensing platforms has gained significant interest in recent years.