A new solid-phase synthesis is shown to be effective in the preparation of cyanine dye Thiazole Orange (TO) and its derivatives, which can be obtained as a traceless cleavage of the Merrifield resin method. The influence of different solvents, substitutional groups, introduced to benzothiazole such as chlorine, methyl, and nitro was extensively studied. The changes of the special characteristic and fluorescence intensity of the TO derivatives were described. The phenomenon of the synergetic effect was also depicted after modifying the TO molecule with chitosan oligosaccharide (CTS) at the end of the alkyl, which can effectively improve the sensitivity of the fluorescent probe.