A rapid, novel and cost‐effective spectrofluorimetric method developed to determine moxifloxacin (MFX) in pharmaceutical preparations because MFX in a pH 10 medium could reduce the fluorescence intensity of l‐tryptophan. The maximum fluorescence excitation and emission wavelengths were found to be 280 and 363 nm respectively. A range of factors affecting fluorescence quenching and the effect of co‐existing substances were investigated. Fluorescence quenching values (ΔF = FL‐tryptophan − FMoxi‐L‐tryptophan) displayed a strong linear relationship with the MFX concentration ranging from 0.2 to 8.0 μg/ml under optimum conditions. The limit of detection was found to be 6.1 × 10−4 μg/ml. The proposed method was shown to be suitable for MFX determination in pharmaceutical tablets and biological fluids by the linearity, recovery and limit of detection. The spectrofluorimetric approach that has been developed is extremely eco‐friendly, as evidenced by the fact that all the experimental components and solvents were safe for the environment.