Graphene oxide (GO) functionalization has great importance for its practical application in many fields, and is a key step for GO bioconjugation. Polydispersity, limited colloidal stability and changeable extinction of GO complicates functional group quantification and the modification study. To bypass the mentioned limitations, we have used GO immobilization on glass support to quantify its epoxy groups surface load, and to study sustainability of the functional groups introduced through epoxy-amine reaction during hydrazine reduction. It was found out that GO contains near 1 epoxy group per nm2, and the major part of the functional groups are remaining after reduction process allowing to use this modification to make functionalized reduced graphene oxide (rGO). Then we modified colloidal GO with azide groups by aminotetraethylene glycol azide (H2N–TEG–N3) in mild conditions to avoid aggregation, and characterized it by means of immobilization on glass surface. Functional group surface load of the functionalized GO was determined as 0,7 groups per nm2. Finally, we proved capability of azide-functionalized GO for conjugation by strain promoted [3 + 2] azide-alkyne cycloaddition (SPAAC) with BCN derivative of organic fluorescent dye JOE.