Nanoparticle-assembled hydroxyapatite (HA) hollow microspheres have a high surface area and are convenient to handle, owing to their characteristic structure. In this study we characterized the protein adsorption of HA hollow microspheres prepared from CaCl 2 and K 2 HPO 4 by a water-in-oil-in-water (W/O/W) emulsion method assisted by two surfactants: Span 80 and Tween 20. The HA hollow microspheres adsorbed bovine serum albumin, bovine γ-globulin, equine skeletal muscle myoglobin, and chicken egg white lysozyme in 10 mM sodium phosphate buffer (pH 6.8) in a Langmuir-type adsorption and desorbed the proteins in 800 mM sodium phosphate buffer (pH 6.8). The maximum adsorbed amounts of the HA hollow microspheres were 7.5–9.0 times higher than those of the microrods with a similar size range. The composite membranes of the HA microspheres and the poly( l -lactic acid) (PLLA) microporous membranes exhibited a high adsorption capacity for γ-globulin.