Agarose hydrogel microspheres imprinted with bovine serum albumin (BSA) were prepared by inverse suspension gelating method. The experimental conditions related with beads' shape and uniformity were studied, including temperature, surfactant content, and stirring speed. The size and distribution of the pores were found related with porogen and surfactant content. Rebinding properties were influenced by the diameter and porosity. The beads with the smallest diameter exhibited the greatest rebinding capacity, rebinding speed, and imprinting efficiency (IE). Smaller pores provided higher rebinding capacity and IE, but lower rebinding speed. Imprinted microspheres showed good specificity toward templating protein compared to others.