Objective To illustrate the interaction process and mechanism of chemokine CCL11 with receptor CCR3 and glycosaminoglycans (GAGs) and shade light on the regulation mechanism of CCL11-GAGs-CCR3.Methods A stably transfected CCR3-EGFP CHO cell line with single-molecule expression level was constructed using genetic engineering. The interaction between GAGs and CCL11 under different in vitro solution was studied by total internal reflection fluorescence (TIRF) and isothermal titration calorimetry (ITC) technique. The regulation of GAGs-CCL11 on chemotactic behavior of CCR3-EGFP stable cells and the effect of CCR3-EGFP aggregation on cell membrane were investigated by chemotactic assay and living cell monomolecular imaging assay.Results With the increase of chondroitin sulfate chain length, the binding heat release of chondroitin and CCL11 increased, indicating that the interaction force was enhanced and the promotion effect of CCL11 aggregation was enhanced. Single molecule fluorescence imaging technology combined with chemotactic test showed that different types and proportions of GAGs could affect the interaction between CCL11 and CCR3. The addition of GAGs inhibited the chemotactic effect of CCL11 on CCR3-EGFP stable cells and promoted the aggregation ability of CCR3-EGFP. With the increase of molecular mass of chondroitin sulfate, the inhibitory effect was significantly enhanced.Conclusion GAGs can affect the aggregation state of CCL11 and its interaction with receptor CCR3. Our study provides a theoretical basis for further elucidating the interaction between CCL11-GAGs-CCR3.