The cyanate anion (CNO-), formed spontaneously within cells from urea and carbamoyl phosphate, usually functions as a biomarker of some diseases such as chronic kidney disease. Therefore, accurate determination of CNO- is highly demanded. Herein, a 3-amino-2-naphthoic acid-based "turn-on" fluorescence probe was developed for specific detection of CNO-. Upon the addition of sodium cyanate, the weak-fluorescent 3-amino-2-naphthoic acid could react with CNO-, which triggered intense emission of green fluorescence. And up to 9-fold fluorescence enhancement was observed. The fluorescence enhancement ratios displayed a good linear relationship with the concentrations of CNO- in the range of 0.5-200 μM. The high selectivity and sensitivity for CNO- detection were investigated with the detection limit as low as 260 nM. The probe was further successfully applied to determine CNO- in real samples such as tap water, human urine and serum samples, which offered a promising approach in practical applications. Graphical abstract.