This study investigates the fluorescence characteristics of formoterol, emphasizing the influence of various experimental conditions on its emission intensity. The fluorescence intensity of FMT was monitored at 342 nm after being excited at 223 nm. Comprehensive optimization of parameters, including pH, buffer solutions, and diluting solvents, revealed that neutral to basic environments and distilled water maximized the fluorescence intensity. The fluorescence behavior of FMT with pH changes was supported using density functional theory (DFT) calculations. Additionally, the change of fluorescence intensity with pH was utilized to calculate the pKa value of FMT. The method was validated per ICH guidelines, demonstrating high linearity, sensitivity, precision, and accuracy. The fluorescence-concentration plot was rectilinear from 50 to 1000 ng mL−1, with high linearity (r2 = 0.9997). The limits of detection and quantitation were 11.5 and 34.9 ng mL−1, respectively. The selectivity of the method was revealed from the absence of any significant interfering effect from the presence of different pharmaceutical excipients. Applications of the developed method in analyzing two commercially available pharmaceutical formulations showed high recovery percentages (99.18 ± 1.72 and 100.00 ± 0. 89). A comparative analysis with existing methodologies revealed superior sensitivity and cost-effectiveness, whereas the greenness evaluation confirmed its environmental friendliness.