Objective:To construct an aggregation induced emission (AIE) self-assembled probe based on glutathione (GSH) response covalent cyclization and evaluate it in vitro.Methods:The peptide sequence containing the 2-cyano-6-aminobenzothiazole-cysteine (CBT-Cys) condensation sequence was synthesized by the solid-phase peptide synthesis method. After coupling with an AIE molecule by click chemical reaction, an AIE self-assembled probe 1 based on GSH response covalent cyclization was constructed, and probe 2 lacking Cys structure was used as the control. The absorption and emission spectra of probes were tested and the specificity of probes to GSH was analyzed. The hydrodynamic diameter and structure of the probes after response were compared. The effects of different pH values, temperatures, probe concentrations, and GSH concentrations on fluorescence intensity were investigated. The toxicity of probes to tumor cells such as HeLa, HepG2 and MDA-MB-231 was evaluated.Results:After GSH response, the fluorescence of probe 1 was enhanced by about 6 times and that of probe 2 was enhanced by about 2 times; probe 1 was converted into a dimer with a hydrodynamic diameter of about 896.1 nm. Probe 2 lacked a cyclization motif and was converted into a monomer with a hydrodynamic diameter of about 427.4 nm. The fluorescence intensity of probe 1 was significantly higher than that of probe 2 at pH=7.0 and 37 ℃, and the toxicity of probes to tumor cells (HeLa, HepG2 and MDA-MB-231) was low.Conclusions:After the disulfide bond of probe 1 was reduced by GSH, the probe molecule lost the hydrophilic sequence, resulting in fluorescence turn-on (the first aggregation), and probe 1 immediately generates an AIE dimer (the second aggregation) because it contains a CBT-Cys cyclization sequence, which realizes the dual AIE effect compared with the single aggregation of probe 2, and significantly enhances the fluorescence emission. Probe 1 has better applicability in physiological environments, which provides an idea for in-situ generation of covalent cycling probes in vivo and is expected to be used in tumor imaging and treatment in the later stages.