The application relates to the field of preparation of fluorescein probes with specific selectivity, and specifically discloses a preparation method of fluorescein probes with specific selectivity. The method comprises: (1) mixing 4-nitrophthalic anhydride, resorcinol, nitrocyclohexane and anhydrous cobalt chloride, heating and melting, adding zinc chloride, reacting, and then adding hydrochloric acid Reflux, filter and wash, dry; (2) will contain Na 2 S and Na 2 SO 3 The reducing solution is mixed with the product of step (1), reacted, and then glacial acetic acid is added dropwise until the precipitation is complete, filtered, washed, and dried; (3) the mixed solvent containing methanol and triethylamine is mixed with the product of step (2) Mix, add CS dropwise 2 , to react, then add copper chloride aqueous solution dropwise, continue the reaction after dropping, then adjust the pH value of the system to 1-2 with hydrochloric acid, distill under reduced pressure, wash, dry, and purify by column chromatography. The method greatly improves the conversion rate and yield of 5-fluorescein isothiocyanate.