The last decade has witnessed tremendous advances in the use of graphene and graphene-based nanocomposites for the fabrication of electrochemical (EC) sensors and biosensors with improved analytical performance. With unique and highly desirable morphology, chemical/thermal stability and EC properties, the graphene-based materials are paving way to the implementation of mediatorless EC detection schemes with direct electron transfer. This approach enables the development of highly performed biosensors with respect to detection sensitivity, precision, specificity, and stability. This review provides a comprehensive overview of the field apart from providing intensive information of the fabrication, properties, characterization and EC applications of graphene and its nanocomposites. Two key challenges, the lack of international regulatory guidelines for nanotoxicity analysis and potential mass production of analytical devices, will also be discussed along with the trends in nanobiotechnology and the requirements in healthcare and industrial applications.