Herein, a novel and rapid fluorometric nanoprobe was constructed for quantitation of dopamine (DA) in presence of biologically interfering compounds. The nanoprobe based on synthesis of yellow emissive nitrogen doped graphene quantum dots (N@GQDs) by advanced thermal driven oxidation. After that, the synthesized N@GQDs was capped with β-cyclodextrin (β-CD), followed by interaction with pyridoxal (PYL) vitamin B6 cofactor. This interaction resulted in diminishing the yellow fluorescence of β-CD/N@GQDs, and appearance of blue emission peak at 420 nm. Upon addition of DA, the blue emission of β-CD/N@GQDs was increased after excitation at λ = 330 nm. Under optimum conditions, the nanoprobe exhibited a linear range of 0.36–400 nM with limit of detection (LOD) of 0.117 nM. In addition, the fluorescent nanoprobe shows high selectivity and can be used for detection of DA in complicated biological matrices and human serum. This strategy might provide a potential tool for clinical diagnosis and biomedical research for DA related diseases.