A simple, precise and accurate method was developed and validated for the determination of allicin release from alliin/alliinase double-layer tablets. According to Appendix XC II of Chinese Pharmacopoeia 2010 edition Volume II, a small glass-method was adopted at the rotational speed of 100 r/min using 100 mL phosphate buffer (pH 6.8) as release medium. The release amount was determined by HPLC with a C18 column (250 mm×4.6 mm, 5 μm) using the mobile phase consisting of methanol -0.4% carboxylic acid (65:35) at a flow rate of 1 mL/min and UV detection at 242 nm. The current method demonstrates good linearity over the range 4.052-405.2 μg/mL (r2=0.9999) with an average recovery of 105.5%(RSD=1.25%). The accumulative release of alliin/alliinase double-layer tablets had good homogeneity for within- and between-batches. The method established is simple, accurate and repeatable for the determination of allicin release from alliin/alliinase double-layer tablets.