The first hyperpolarizability of graphene quantum dots (GQDs) suspended in water was determined using the hyper-Rayleigh scattering (HRS) technique. To the best of our knowledge, this is the first application of the HRS technique to characterize GQDs. Two commercial GQDs (Acqua-Cyan and Acqua-Green) with different compositions were studied. The HRS experiments were performed with an excitation laser at 1064 nm. The measured hyperpolarizabilities were (1.0±0.1) × 10^(-27) esu and (0.9±0.1) × 10^(-27) esu for Acqua-Cyan and Acqua-Green, respectively. The results were used to estimate the hyperpolarizability per nanosheet obtained by assuming that each GQDs has five nanosheets with 0.3 nm thickness. The two-level model, used to calculate the static hyperpolarizability per nanosheet, provides the values of (2.4±0.1) × 10^(-28) esu (Acqua-Cyan) and (0.5±0.1) × 10^(-28) esu (Aqua-Green). The origin of the nonlinearity is discussed on the basis of polarized resolved HRS experiments, and an electric quadrupolar behavior with a strong dependence on surface effects is found. The nontoxic characteristics and order of magnitude indicate that these GQDs may be useful for biological microscopy imaging.