Surface functionalization of nanomaterials with specific recognition units is a promising strategy to structure novel nanosensors for target analysis and bioimaging. In this study, a fluorescent nanosensor has been successfully synthesized by conjugating thiosemicarbazide with carbon dots (CDs) through amide bonds. The functional CDs own a relatively narrow size distribution of about 1.6-4.0 nm. The CDs exhibit highly specific recognition capability towards copper ions (Cu 2+ ) over the other competing metal ions, and the maximum quenching rate reaches to 68.7%. A good linear correlation is also established between F0/F and the Cu 2+ concentrations ranging from 0 to 0.4 µM and the detection limit is 3.47 nM. On account of their strong fluorescence intensity, excellent water solubility, low toxicity and good biocompatibility the nanosensor demonstrates their biolabeling potential in vivo, which might be high significance in bioanalysis and biomedical detection in the future.