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With the wide application of nanomaterials, the quantification of functional groups on nanomaterial surface becomes more and more necessary. A heterogeneous 9-fluorenylmethoxy carbonyl chloride (Fmoc-Cl) fluorescent method using an aqueous solution was established to determinate amino groups on nanomaterial surface. The effect factors of determination were investigated and the assay was optimised. The Fmoc fluorescent method is 200-fold more sensitive than the current UV assay using an organic solvent, and compared with chemical ninhydrin method and physical elemental analysis. Heterogeneous Fmoc-Cl fluorescent method can be used to determine amino groups on nanomaterials with big size, which is difficult to undergo a direct detection.