To develop a RP-HPLC method for determination of vitamin D₃ in calcium tablet with microwave extraction. Methods Vitamin D3 was extracted by microwave in the ethanol. The HPLC analysis was carried out on a C18 column (4.6 mm×150 mm,5 μm) with ethanol at the flow rate of 1 ml/min. The detection wavelength was set at 264 nm. Results The calibration curves were linear within the range of 0-4 μg/L (r=0.999 8) , and the detection limit was 0.02 μg/L.Conclusion The recoveries for vitamin D3 were between 98.8%-100.0%, and RSD was 0.4%. This method was simple, accurate and practical for the detection of vitamin D₃ in health food.