A facile two-step method for preparing chitosan-based immobilized metal ion affinity chromatography was developed. First, chitosan was phosphorylated by esterification with phosphoric acid, and then titanium was chelated onto the phosphorylated chitosan. The obtained chitosan-based titanium immobilized metal ion affinity chromatography was ultrafine microparticles and had good dispersibility in acidic buffer. The selectivity and sensitivity were evaluated by phosphopeptide enrichment of mixtures of α-casein and bovine serum albumin. The enriched peptides were analyzed by mass spectrum. Enrichment protocols were optimized and the optimum-loading buffer was 80% acetonitrile with 1% trifluoroacetic acid. With α-casein concentration as low as 2 pmol, 12 phosphopeptides were detected with considerably high intensity from the digest mixtures of α-casein and bovine serum albumin with molar ratio of 1:200. The microparticles was also applied in real biological samples, 29 phosphoproteins containing 40 phosphorylated sites were identified from salt-stressed Arabidopsis thaliana leaves.