Abstract: In order to expand the application of natural vanillin, the crude product of vanillin glycoside was extracted from vanilla beans by ultrasonic assisted solvent extraction. The extraction process was optimized by a single factor test and orthogonal test with extraction rate as the index, and vanillin glucoside monomer was separated by high performance liquid chromatography. The results showed that: 1) The optimal extraction conditions for the crude product were: 10 min extraction time, 65 ℃ extraction temperature, a solid to liquid ratio (mass ratio) 1:11, and 140 W extraction power. Under these conditions, the extraction rate of vanillin glucoside was 1.88%. 2) When the ratio of acetic acid aqueous solution (mass percentage 0.5%) to methanol in the mobile phase was 80:20 (v/v), vanillin glucoside monomer was effectively separated by the preparation high performance liquid chromatography with a product yield of 1.36%. 3) The structure of vanillin glycoside was characterized by ¹H NMR and ¹³C NMR, which confirmed that the obtained product was vanillin glucoside. The extraction and separation processes had the advantages of a high processing capacity, simpler steps and lower cost, thus providing a reference for the development of natural vanillin glucoside.