A new method for determination of uridine, guanosine and adenosine contents in Allium macrostemon bunge were established by using RP-HPLC gradient elution. The extracts were separated on a ZORBAX-SB-C18 analytical column (4.60 mm×250 mm, 5 μm) with CH3OH-H2O as mobile phase at 260 nm. The flow rate was 0.6 mL?min- 1and the column temperature was 25 ℃. The linear ranges of uridine,guanosine and adenosine were 0.0784~0.784 μg（r=0.9995）, 0.0798~0.798 μg（r=0.9995）and 0.0852~0.852 μg (r=0.9994), respectively. This method showed high reproducibility and can be used for quality control of the Allium macrostemon bunge.