The invention discloses a pair of isomers, and a preparation method and application thereof, and the chemical structures of the pair of isomers are shown as formulas (I) and (II): the preparation method comprises the following steps: mixing (E)-2-(2-(Z)-1, 3, 3-trimethylindole-2-ethylene) ethylene) furan [2, 3-b] quinoline-3, 4 (2H, 9H)-diketone, diethyl sulfate, anhydrous potassium carbonate andan organic solvent, performing heating and reacting, concentrating an organic phase, and performing separating to obtain a product. According to the invention, two compounds are prepared by adopting aone-pot method, and the small organic molecule (I) can realize positioning and fluorescence response to lipid droplets in cells, and has the advantages of high sensitivity, rapid response, high specificity and the like; the small organic molecule (II) has fluorescence response to solution viscosity, can be used for detecting system viscosity, has strong acetylcholin esterase inhibition activity and can be used for treating related diseases.