Graphene nanosheet hybridized with polydiacetylene (PDA) can be used as the electrochemical biosensor applications. Self-assembly and photo-polymerization chemistry of diacetylenic monomers on reduced graphene oxide (RGO) for non-covalent functionalization were used for high solubility and further functionality of graphene in water necessary for biosensor application. This enables regular graphene micropatterns to be formed on large substrates with chemical bonding and biological interactions. The PDA/RGO nanohybrids demonstrated the formation of conjugated polymer on graphene for fluorescence and electrochemical detection. The optical and structural properties of PDA/RGO, characterized by UV-Vis, FT-IR, Raman, and photoluminescence spectrometers, demonstrated that PDA and RGO in PDA/RGO are connected by physical interactions. Further-functionalization action of PDA/RGO with biotin was verified in microfluidic biochip via biotin-straptavidin interaction. Here, the applicability of functionalized RGO in the biochip and biosensing platform based on fluorescence detection was confirmed. The electrochemical experiments showed the potential of functionalized RGO for use as an electrochemical biosensor, especially with the detection of an analyte at low concentration.