With the increasing requirement for analysis and separation of samples related to genomics, proteomics, metabolomics, pharmacology and agrochemistry, diverse stationary phases for liquid chromatography have been prepared by Cu(i)-catalyzed 1, 3-dipolar azide-alkyne cycloaddition reaction (CuAAC). It has been proved that CuAAC is a powerful tool for preparing covalently bonded stationary phases. In this tutorial review, we highlighted the preparation of separation materials by immobilization of functional groups on silica beads, polymer beads and agarose via CuAAC and their applications in liquid chromatography and related purposes, such as separation of polar compounds, enrichment of valuable bio-samples, orthogonal two-dimensional HPLC and chiral separation. Meanwhile, agarose-based separation materials for affinity chromatography are reviewed.