An acid-treated, oxidized graphitic carbon nitride nanosheets (g-C 3 N 4 NSs) was successfully synthesized and applied as a fluorescence probe for the detection of hemin based on the inner filter effect (IFE). The results indicated that the fluorescence quenching of g-C 3 N 4 NSs caused by hemin was attributed to the overlap between absorption spectrum of hemin and emission spectrum of g-C 3 N 4 NSs. It is confirmed that the g-C 3 N 4 NSs exhibited a highly sensitive and selective for detection of hemin. Under the optical condition, the fluorescence changing of g-C 3 N 4 NSs is related to the concentration of hemin ranging from 0.5 -25 μM with a detection limit of 0.15 μM. Moreover, the fluorescence probe has been successfully applied to detect hemin in human blood. Overall, a novel and efficient fluorescence-based method was developed supported by g-C 3 N 4 NSs, which is a promising material for application in sensing and cell imaging.