<p>A novel green spectrofluorimetric technique was developed for the quantitative analysis of moxifloxacin (MOXI) in pure form, commercial formulations, and biological samples utilising deep eutectic solvent (DES) instead of an aqueous medium. Ethaline, a deep eutectic solvent composed of choline chloride and ethylene glycol, was utilised as the environmentally friendly solvent system. The approach exhibited enhanced analytical performance compared to water-based analysis, evidenced by a 1.2-fold increase in fluorescence intensity and a blue shift in both excitation and emission wavelengths. The calibration graph demonstrated a linear association between fluorescence intensity and moxifloxacin concentrations ranging from 0.2 to 10&#xa0;µg mL^− 1, with a correlation coefficient of 0.9993. The detection and quantification limits were established at 0.0165&#xa0;µg mL^− 1 and 0.0495&#xa0;µg mL^− 1, respectively. The influence of standard excipients was examined, although no interferences were identified. The established approach has been employed to study standard forms of moxifloxacin, commercial formulations, and biological samples. The approach, which effectively adhered to ICH standards, was employed for the examination of moxifloxacin in its pure state, pharmaceutical formulations, and biological specimens. The recovery percentages varied from 99.68% to 101.060% for pharmaceutical preparations and from 98.77% to 101.230% for human blood plasma and urine. This green analytical procedure can be used as a viable and green alternative for the routine pharmaceutical analysis with better sensitivity.</p>