A green spectrofluorimetric method for determining Bilastine (BIL) was described. The method is very sensitive, simple, and quick. The suggested method was depend on the measurement of the original fluorescence of BIL in 1.0 M sulfuric acid at an emission wavelength of 385 nm after an excitation at 272 nm. The method was evaluated by ICH requirements. The relationship between BIL concentrations and the fluorescence intensities was linear in a range of 10.0–500.0 ng mL− 1, and the correlation coefficient was 0.9999. The detection limit was 2.9 ng mL− 1 and quantitation limit was 8.8 ng mL− 1. The suitable sensitivity and selectivity of the suggested method enabled its application successfully in analyzing BIL in real human plasma with appropriate recoveries from 95.72% to 97.2 %, as well as in pharmaceutical preparations without any interfering effect from plasma components or pharmaceutical excipients. Additionally, the suggested method was utilized efficiently for content uniformity test.