A novel and sensitive spectrophotometric method is developed for the determination of nanomolar level of nile blue A in water samples based on its catalytic effect of silver nanoparticles on the oxidation of nile blue A by hexacyanoferrate (III) in acetate-acetic acid medium and at 25 ℃. The absorbance is measured at 595.5 nm with the fixed-time method. The optimization of the operating conditions regarding concentration of the reagents, temperature and interferences is also investigated. The calibration curve is linear over the concentration range between 85 and 2000 nM of nile blue A with good precision and accuracy. The detection limit of the method is down to 54. The relative standard deviation for a standard solution of 100.0 nM of nile blue A is 1.63% (n = 10). The proposed method provides a highly sensitive, selective and relatively rapid assay for nile blue A at nanomolar level without any pre-concentration and separation step. The method was applied to the determination of nile blue A in river water samples.