While much effort has been made to prepare magnetic microspheres (MMs) with surface moieties that bind to affinity tags or fusion partners of interest in the recombinant proteins, it remains a challenge to develop a generic platform that is capable of incorporating a variety of capture ligands by a simple chemistry. Herein, we developed core-shell structured, magnetic microspheres with a high magnetic susceptibility and a low non-specific protein adsorption. Surface functionalization of these MMs with azide groups facilitates covalent attachment of alkynylated ligands on their surfaces by "click" chemistry, and creates a versatile platform for selective purification and immobilization of recombinant proteins carrying corresponding affinity tags. The general applicability of the approach has been demonstrated in incorporating four widely used affinity ligands with different reactive groups (-CHO, -SH, -COOH, and -NH2) onto the MMs platform for purification and immobilization of targeted proteins. The azide-functionalized MMs would be applicable for a variety of ligands and substrates that are amenable to alkynylation modification.