Due to the growing need for sensitive, reliable, reusable, fast, and cheap devices for the detection of analytes which have an important role in diagnosis of different diseases, in metabolic disorders, in monitoring treatment of serious diseases such as cancers, the sensing field has attracted huge interest from the scientists. The majority of the traditional methods that are currently in use are invasive, expensive, and laborious. Moreover, highly specialized operators and sophisticated instrumentations are usually required. Taking these into account, the introduction of electrochemical sensors and biosensors avoid a lot of the disadvantages associated with most of the used analytical techniques. The biggest contribution to this development was the use of different nanomaterials as transducers of the analytical signal. The properties, such as high mechanical strength, good electrical conductivity, and ability to serve as efficient signal transducers, make carbon-based nanomaterials, including graphene, ideal materials for biosensor applications. Furthermore, graphene presents high surface area that can be easily modified in different ways to be adapted for the immobilization of various biocompounds for the construction of biosensors. Recent advances regarding the use of graphene and graphene materials for the immobilization of several enzymes for biosensor development and their applications for the detection of chemical and biological species are presented with focus on different enzymes immobilization techniques. In the end, the future trends for the development of graphene-based biosensors in biomedical field are also discussed.