•Investigate adsorption equilibriums and mass transfer processes of monosaccharides.•Optimize operational conditions to separate monosaccharides in biomass hydrolysate.•Arabinose and xylose were recovered through cation resin chromatography. Recovery of monosaccharides from hydrolysates of biomass wastes can bring environmental and economic benefits. This study aimed to explore the feasibility to separate major monosaccharides from hydrolysates of lignocellulosic biomass, i.e. glucose, xylose and arabinose by using column chromatography process. Cation exchange resin Amberlite IR120 and Amberlite IRP69 in Na+ and Ca2+ forms were chosen as adsorbents. The adsorption behaviors were firstly inspected by adsorption equilibrium and mass transfer coefficients. Then the optimal chromatography conditions were determined by using synthetic solution. Finally, hydrothermal liquefaction hydrolysate of pine branches was separated. The results showed that cation resin Amberlite IRP69 (Ca2+) had high adsorption selectivity for arabinose which could be recovered from synthetic solution with purity of 92%. The highest purity of 88% was achieved for xylose when using this resin to separate pretreated hydrolysate. This work suggests that cation exchange resin could be further developed for effective separation of monosaccharides mixture.