In this study, a dual-channel probe of FlE based on flavonoid derivatives is reported, which can yield “turn-on” signals of both fluorometry and ¹⁹F nuclear magnetic resonance in response to the presence of esterase. Upon the addition of esterase, FlE could convert into Fl, possessing the ESIPT effect. As a result, the fluorescence intensity was significantly enhanced and peaked at 510 nm, accompanying a change in the fluorescence of solution from nonluminous to strong green. Moreover, as determined by ¹⁹F NMR, the signal could apparently shift from δ_F −111.57 to −111.69 ppm. Due to the combination of these two detection approaches having good sensitivity, high selectivity, and real-time detection, FlE has been successfully applied to confocal fluorescence imaging for the detection of esterase in cells, which consistent with the ¹⁹F NMR test results in some sense.