To determine brassinolide in rice(Oriza sutiva L.) using HPLC equipped with fluoresence detector, a highly sensitive fluorescence reagent. 1-cyanoisoindole-2-m-phenylboronic acid, was synthesized from the reaction of o-phthaldehyde, m-phenylboronic acid and KCN, then was reacted with brassinolide. The formation ratio of brassinolide boronate exhibited 90% up at the ratio of 20 : 1(㎍/㎍) of 1-cyanoisoindole-2-m-phenylboronic acid and brassinolide respectively. The detection limit of brassinolide boronate with fluoresence detector was 0.16 ng. Brassinolide was detected in heading stage(biomass : 10 g) and panicle formation stage(biomass : 100 g) of the rice(Oryza sativa L.) with quantity of 0.8 ㎍ and 0.2 ㎍ respectively. However, brassinolide was not detected in blooming and elongation stage.