Many non-invasive novel nano-formulations were used for delivery from the ocular surface to posterior segment of the eye, while dexamethasone (DEX) ternary cyclodextrin (CD) aggregate suspension eye drops had recently entered phase III clinical trial. In this study, DEX quaternary CD aggregate suspension eye drops containing HPγCD as solubilizing CD, γCD as aggregating CD and poloxamer 407 were developed and easy for large-scale production. The permeability of DEX quaternary CD aggregate (DEX/γCD/HPγCD/P407) eye drops was better than that of DEX ternary CD (DEX/γCD/P407) eye drops in human corneal epithelial cells and human conjunctival epithelial cells. Ocular distribution revealed that the concentrations of DEX in the choroid-retina were still higher than the effective therapeutic concentration within12 h. DEX/γCD/HPγCD/P407 eye drops remained stable for 6 months in long-term testing and accelerated testing. In conclusion, DEX quaternary CD aggregate eye drops offer an efficient strategy for drug delivery from the ocular surface to posterior segment of the eye.