In this article, chitosan-hydroxyproline resin microspheres (CHRM) were used as adsorbent for the separation of hydroxyproline (Hyp) from mixed amino acids. The optimum operating conditions (e.g., concentration of Hyp, pH of solution, and the optimum eluting agent) were determined by batch adsorption and elution of Hyp on CHRM. Under the optimum operating conditions, the effect of the flow rate of Hyp solution on dynamic adsorption and elution was studied. Furthermore, the selective adsorption property of CHRM for Hyp was investigated. The results indicate that Hyp can be effectively separated from mixed amino acids using CHRM as adsorbent. The hydrophobic interaction between the Hyp and CHRM play an important role in the selective adsorption.