Ultrathin graphitic carbon nitride (g-C3N4) nanosheets, due to their interesting two-dimensional graphene-like structure and unique physicochemical properties, have attracted great research attention recently. Here, a new approachis developed to prepare, for the first time, proton-functionalized ultrathin g-C3N4 nanosheets by sonication-exfoliation of bulk g-C3N4 under an acid condition. This method not only reduces the exfoliation time from more than 10 h to 2 h, but also endows the nanosheets with positive charges. Besides retaining the properties of g-C3N4, the obtained nanosheets with the thickness of 2-4 nm (i.e., 6-12 atomic monolayers) also exhibit large specific surface area of 305 m2 g-1, enhanced fluorescence intensity, and excellent water dispersion stability due to their surface protonation and ultrathin morphology. The well-dispersed protonated g-C 3N4 nanosheets are able to interact with negatively charged heparin, which results in the quenching of g-C3N4 fluorescence. A highly sensitive and highly selective heparin sensing platform based on protonated g-C3N4 nanosheets is established. This metal-free and fluorophore label-free system can reach the lowest heparin detection limit of 18 ng mL-1. Proton-functionalized g-C 3N4 ultrathin nanosheets with positively charged surface are synthesized by liquid exfoliating acidified bulk g-C3N 4. The obtained g-C3N4 nanosheets with 6-12 atomic monolayers and large surface area up to 305 m2 g-1 exhibit outstanding sensitivity and selectivity in the fluorescence-quenching sensing of heparin under both ideal and physiological conditions.