A spectrofluorometric method was developed for determination of brassinosteroids plant hormones based on the reaction between brassinolide and m-dansylaminophenylboronic acid. 90 μM of m-dansylaminophenylboronic acid solution was chosen as the optimal concentration of the reagent. The temperature of the reaction was 30°C (room temperature) for 20 min duration in 5 mM phosphate buffer solution, pH 7. The excitation and emission wavelengths were 380 nm and 505 nm, respectively. Under optimal condition, linearity was found in the range 1-50 μM. Standard addition was used for the brassinosteroids determination in bio-extract samples, without any sample preparation. Limit of detection (3SD, n=10) of 0.24 μM and limit of quantitation (10SD, n=10) of 0.48 μM were obtained using sample blank with this standard addition method. Result was demonstrated to be a suitable semi-quantitative technique and was applied successfully for the determination of total brassinosteroids plant hormones equivalent to brassinolide.