Hydroxyapatite (HAP) is a common bio-adsorbent, which performance depends heavily upon its morphology and microporous structure. In this study, a novel synthesis strategy was proposed for hierarchical porous HAP microspheres by a simple “one-pot” hydrothermal reaction. In the strategy, L-glutamic acid serves as soft template to modulate the morphology and inner crystalline of HAP. To evaluate the application potential, doping Ni 2+ on hierarchical porous HAP microspheres gives metal chelated affinity adsorbents. The prepared adsorbents show a perfect spherical shape, particles size of 96.6 μm, relatively specific surface area of 48.5 m 2 ·g −1 and hierarchical pores (mesopores: 4 nm and macropores: 53 nm). By the adsorption evaluation, it reveals that the Ni 2+ -HAP adsorbents have high adsorption capacities of 275.11 and 97.55 m 2 ·g −1 for hemoglobin and bovine serum albumin, respectively, which is comparable to other similar adsorbent. Therefore, this work provides a promising method for high-efficiency hydroxyapatite microspheres for proteins purification.