A novel method for the preparation of gelatin sponge millispheres (GSMs) for biomaterials such as embolic agents and cell scaffolds was developed using an air-in-water-in-oil-type emulsion. The droplets, consisting of a foamy gelatin suspension in caprylic triglyceride, were gelled and rinsed with isopropanol. Sonication and depressurization were used during the rinsing process to create interconnected pores. GSMs cross-links created over 4 h at 155°C without any agent were insoluble and had short and long diameters of 1.1 ± 0.2 mm and 1.3 ± 0.2 mm, respectively. The residual isopropanol and caprylic triglyceride were <0.05% (w/w) and <1% (w/w) respectively. The level of bacterial endotoxins in the extracts was below 0.025 EU/ml, and no bacterial or fungal growth was found during sterility testing. The GSMs produced using this method were considered to meet the basic requirements of embolic agents.