Graphene oxide (GO), the oxidized form of graphene, shows unique properties such as strong mechanical strength, high electrical and thermal conductivity, amphiphilicity and surface functionalization capability that make it very attractive in various fields ranging from medicine to optoelectronic devices and solar cells. However, its insolubility in non-polar and polar aprotic solvents hinders some applications. To solve this issue, novel functionalization strategies are pursued. In this regard, the current study deals with the preparation and characterization of hexamethylene diisocyanate (HDI)-functionalized GO. Different reactions conditions were tested to optimize the functionalization degree (FD), and detailed characterizations were conducted via elemental analysis, Fourier-transformed infrared (FT-IR) and Raman spectroscopies to confirm the success of the functionalization reaction. The morphology of HDI-GO was investigated by transmission electron microscopy (TEM), which revealed an increase in the flake thickness with increasing FD. The HDI-GO showed a more hydrophobic nature than pristine GO and could be suspended in polar aprotic solvents such as N,N-dimethylformamide (DMF), N-methylpyrrolidone (NMP) and dimethyl sulfoxide (DMSO) as well as in some low polar/non-polar solvents like tetrahydrofuran (THF), chloroform and toluene; further, the dispersibility improved upon increasing FD. Thermogravimetric analysis (TGA) results confirmed that the covalent attachment of HDI greatly improves the thermal stability of GO, ascribed to the crosslinking between adjacent sheets, which is interesting for a variety of applications including long-term electronics and electrothermal devices. The HDI-GO could further react with other organic molecules or polymers via the remaining oxygen groups, which makes them ideal candidates as nanofillers for high-performance GO-based polymer nanocomposites.