The emergence of graphene (GR) has recently opened up an exciting new field in the science and technology of two-dimensional (2D) nanomaterials with continuously growing academic and technological impetus. GR exhibits unique electronic, optical, magnetic, thermal, and mechanical properties arising from its strictly 2D structure and, thus, has many important technical applications. In the last 2 years, GR, as novel sensing material has attracted tremendous attention and research interest. To further improve electrochemical property, large numbers of GR-based hybride materials have been well designed, synthesized, and investigated for sensing application. The hybridization can be an effective strategy to enhance functionality of the materials, and the integration of nanomaterials on GR potentially paves a new way to enhance their electronic, chemical, and electrochemical properties. As a result, the GR composites often offer better analytical characteristics than GR materials alone. However, the immobilizations of GR-based material and enzyme are important to enhance electrochemical properties and use of the biosensor. Classical coating method results in poor stability due to loss of the enzyme/antibody. To resolve the problem, we developed a green and controllable strategy to fabricate well-dispersed GR-metal nanocomposite modified functional conducting polymer film containing carbonyl groups with electrochemical deposition. Horseradish peroxidase was finally connected covalently to the film with 1-ethyl-3-(3-dimethylaminepropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS) as activator. The as-prepared GR/gold nanocomposite offers remarkable catalysis performance to the redox of hydrogen peroxide on the electrode surface. In this review, from the viewpoint of chemistry and materials, we will cover recent significant advances in hybrids, design, and synthesis of GR-based film, as well as immobilization of enzyme/antibody on the electrode, and analytical application together with discussion on the major challenges and opportunities for future GR research.