The spherical chitosan adsorbent was prepared by graft copolymerization of acrylic acid onto the cross-linked spherical chitosan beads, and then was adopted to adsorb the L-histidine. The adsorption conditions, i.e., solution pH, adsorption time, initial concentration and adsorption temperature were optimized. The spherical chitosan adsorbent showed excellent equilibrium adsorption capacity of 78.3 mg/g for the L-histidine when the solution pH value was 7.5, adsorption time was 180 min, initial concentration was 1500 mg/L and the adsorption temperature was 25 °C. Moreover, the inorganic salt of NaCl also showed great effect on the equilibrium adsorption capacity exceeding 0.8 mol/L. The static adsorption processes followed the Langmuir adsorption isothermal equation and Freundlich adsorption isothermal equation. Furthermore, L-histidine could be desorbed with 1.5 mol/L of ammonia solution, and the regeneration capacity of the spherical chitosan adsorbent was excellent.