Since graphene exhibits innovative mechanical, electrical, thermal, and optical properties, this 2D material is increasingly attracting attention and is under active research. Among the various graphene forms with lattice-like nanostructure, graphene oxide (GO) displays advantageous characteristics as a biosensing platform due to its excellent capabilities for direct wiring with biomolecules, a heterogeneous chemical and electronic structure, the possibility to be processed in solution and the ability to be tuned as insulator, semiconductor or semi-metal. Moreover, GO photoluminescences with energy transfer donor/acceptor molecules exposed in a planar surface and is even proposed as a universal highly effi cient long-range quencher, which is opening the way to several unprecedented biosensing strategies. Here, the rationale behind the use of GO in optical biosensing applications is discussed by describing different potentially exploitable properties of GO, and an overview of the current approaches are presented along with future perspectives and challenges.