The method for determination of adenosine in zymolytic powder of aweto was studied. The detecting wavelength, proportion of mobile phase, chromatography mapping character, adenosine recovery ratio and the ultrasonic pretreatment were discussed. Reslutes showed that, using a reverse-phase bonded ODS Hypersil column (5 μm, 125×4 mm), the most suitable mobile phase and detecting wavelength were 0.06 mol/L KH2PO4- methanol-tetrahydrofuran (150:10:1.5) and 260 nm, respectively, under which the average recovery was 99.11%. The method was very sensitive and selective and the pretreatment method was simple and fast.