Previously, we developed polyethylenimine-coated hydroxyapatite as a chromatography medium. This report describes a simple, safe, productive, and improved preparation method and reports further chromatographic properties. The improved polyethylenimine-coated hydroxyapatite was prepared by use of a rotary evaporator with neither decantation, cross-linking agent, nor organic solvent. In chromatography, ovalbumin (a phosphoprotein) was separated from other proteins. Furthermore, ovalbumin derivatives or nucleotides could be separated on the basis of the number of phosphate residues. This method yielded high-quality and -quantity polyethylenimine-coated hydroxyapatite.