A simple and sensitive spectroelectrochemistry method for the determination of vitamin K3 was developed by combining electrolysis and fluoremetry. This method was based on that vitamin K3 was reduced at a glassy carbon electrode, and its product with characteristic fluorescence at 420 nm was determined with excitation wavelength at 309 nm. Under optimized electrochemical reaction conditions and fluorescent experiment parameters, the fluorescence intensity was proportional to the concentration of vitamin K3 in a range from 3.50×10-7 to 1.05×10-5 mol/L with a correlation coefficient of 0.9991, and detection limit was estimated to be 7.50×10-8 mol/L at a signal/noise ratio of 3. The relative standard deviation was less than 4.3%(n=5) and the recovery was in a range of 97%-105% for the determination of vitamin K3 in pharmaceutical preparations. The result is satisfactory for the determination of vitamin K3 as comparison to that from HPLC method.