Rhodamin B is a textile or clothing dye that is often misused for food and beverage dyes. This study aimed to obtain a valid method for the analysis of rhodamine B within red syrup through spectrofluorometric method. The validation of the analysis methods included linearity, LOD, LOQ, precision, and accuracy. In this study, it were used λ 550 nm excitation and λ emission 570 nm. Furthermore, it was obtained CV (Coefficient of Variation) value in precision test was 0.287% ((2%). The average percent recovery value on the accuracy test was 102.68% (80-120%). The linearity was indicated by the correlation coefficient value (r = 0. 99746> r table = 0. 087). The limitation of detection and limit of quantitation obtained from this research were 0.14 ppm and 0.47 ppm. Based on the validation result, the spectrofluorometric method was valid and the method of sample red syrup was not found rhodamin B.