Reduced graphene oxide (rGO) is widely utilised to develop various types of biosensors; however, producing self-assembled rGO nanoflake networks through single-droplet drop-casting remains inconsistent. In the present work, we systematically used three different methods to prepare rGO suspensions in order to produce large scale self-assembled rGO nanoflake networks through single-droplet drop-casting. The rGO suspensions were prepared using only deionised water with no added any chemicals/organic solvents, which we considered to be a low-cost method. Subsequently, the most effective preparation method was used to deposit rGO nanoflakes onto commercial gold interdigitated microelectrodes (Au-IDE) to examine their electrical performance. Assessment of the yields, developed methods, surface morphologies, spectroscopy and structural analyses of the as-prepared rGO nanoflakes were conducted. The results revealed that method-3 (involving sonication, centrifugation and post-sonication) produced large self-assembled rGO nanoflake networks with strong adhesion to glass substrates. Furthermore, the as-prepared rGO/Au-IDE modified sensors showed excellent electron mobility where the electrical conductivity was enhanced approximately similar to 1000 fold compared to the bare devices. The present work provided new insights for depositing large self-assembled interconnected rGO nanoflake networks through single-droplet drop-casting which will be beneficial for biosensor development and other down- stream applications. (C) 2020 Elsevier Inc. All rights reserved.