The peroxidase-like activity of 2',7'-difluorofluorescein (DFF), was investigated using 3,3',5,5'-tetramethylbenzidine (TMB) as a chromogenic substrate in the presence of H₂O₂. DFF could catalyze oxidization of TMB by H₂O₂ to produce a blue colored oxidation product. Effect of various reaction conditions, such as pH, temperature, H₂O₂ concentration and reaction time on the catalytic activity of DFF was studied. The peroxidase-like activity of DFF was found to follow Michaelis–Menten kinetics, and its catalysis accorded with ping-pong mechanism. The calculated kinetic parameters (K_cat) of DFF catalysis showed higher peroxidase-like activity than fluorescein and 2′,7′-dichlorofluorescein (DCF). According to the radical capture and electron spin resonance (ESR) spectroscopy results, we confirmed that hydroxyl radical (•OH) is the active specie of catalytic process. It is known that the oxidation of galactose by galactose oxidase (GAOx) enzyme leads to the formation of H₂O₂, the H₂O₂ released in this reaction was consequently quantified using DFF as peroxides mimics and TMB as the chromogen. Thus, a combination of above two reactions was exploited to establish a method for galactose detection. Under the optimum conditions, the linear range of this method was from 10 μM to 20 mM with the detection limit down to 3 μM. Moreover, the developed method was applied to detect galactose in urine samples. Our work will facilitate the utilization of DFF intrinsic peroxidase-like activity in medical diagnostics and biotechnology.