In order to enrich the types of chitosan-based chiral stationary phases and meanwhile discover new promising chiral separation materials for efficient enantioseparation, four chloro-substituted chitosan isobutylurea derivatives were prepared and proposed as chiral selectors in this work. The chitosan isobutylurea derivatives were characterized and were found to be successfully synthesized with expected structures. It was observed that the prepared chiral selectors exhibited a low swelling capacity in ethyl acetate, acetone or a mixed solution of n-hexane and a certain proportion of tetrahydrofuran, and the corresponding chiral stationary phases (CSPs) showed good solvent tolerance. These facts indicated that chiral separation of enantiomers could be performed under more types of chromatographic conditions with some “unusual organic solvents” as mobile phase additives. In addition, the prepared CSPs also possessed excellent chiral recognition and enantioseparation ability for the tested chiral samples. Compared with common coated-type CSPs derived from amylose and cellulose, the chitosan-based CSPs prepared in the present study exhibited more preferable enantioseparation performance for some analytes.