The spectrofluorometric method, based on fluorescence properties of aluminium (III)–hesperidin complex, for the determination of hesperidin in human plasma and pharmaceutical forms has been developed and validated. The complex shows strong emission in the presence of surfactant betain sulfonate SB 12 at 476 nm with excitation at 390 nm. Linearity range in pharmaceutical forms of hesperidin was 0.06 – 24.4 μg mL-1 with LOD 0.016 μg mL-1 and LOQ 0.049 μg mL-1. Recovery values in the range 99.3 – 99.7% indicate good accuracy of the method. A linear dependence of the intensity of fluorescence of the complex on the concentration of hesperidin in plasma was obtained in concentration range from 0.1 – 12.2 μg mL-1. The LOD was 0.032 μg mL-1 while LOQ was 0.096 μg mL-1. Recovery values were in the range 98.4 – 99.8%. The reliability of the method was checked by LC-MS/MS method for plasma samples and HPLC/UV method for tablets with direct determination of hesperidin after separation. Linearity range in determination of hesperidin in pharmaceutical forms was obtained in the range from 0.05 to 10.00 μg mL-1. The LOD was 0.01 μg mL-1 and LOQ was 0.03 μg mL-1. Linearity range in plasma determination of hesperidin was 0.02 – 10.00 μg mL-1 with LOD 0.005 μg mL-1 and LOQ 0.015 μg mL-1. Good agreement between two methods indicate the usability of the proposed spectroflurometric method for the simple, precise and accurate determination of hesperidin in clinical and quality control laboratories.