The objective of this study is to develop the process conditions of a w/o emulsification method for the preparation of the smallest agar nanoparticles possible in a low nanometer range with the highest yield, prior to adding an aqueous solution containing an unstable drug such as a protein drug for its encapsulation. The volume ratio between the organic phase and aqueous phase was set as 10 : 1, the lowest ratio among those of previous investigations. Agar solutions of 0.5, 1 and 2%w/v concentration were emulsified in soybean oil. Then, various homogenizer speeds were investigated for the yield of agar nanoparticles and their particle size distribution under those agar concentrations. The visual appearance of the re-dispersed emulsion-converted-to suspension was turbid without any visible aggregates, even though the suggested methodology is more economically efficient in terms of the ratio between the organic phase and aqueous phase, than those of previous investigations. In terms of agar concentration, the yields of agar nanoparticles were optimized at 1%w/v of agar concentration for all the homogenizer speeds of 13000, 15000 and 17000 rpm. Accordingly, the yield of 76.5% was the highest at the preparation conditions of 1%w/v agar concentration and 15000 rpm homogenizer speed.