Di(tridecyl) phosphoric acid (DTDPA) was synthesized as a new surfactant for protein extraction by reversed micelles. DTDPA can form stable reversed micelles in the concentration range above 1 mM. The size of the DTDPA reversed micelles was smaller than that of conventional AOT (sodium-di-2-ethylhexyl sulfosuccinate) reversed micelles under the same conditions. The formation of reversed micelles using DTDPA can be controlled by adjusting the aqueous pH. The extraction behavior of some proteins from an aqueous phase into the reversed micellar phase of DTDPA has been investigated by varying the experimental conditions (pH, ionic strength, surfactant concentration). It was found that reversed micelles with DTDPA can extract many proteins. In particular, hemoglobin, which is difficult to extract with an AOT reversed micelle, can be extracted in the DTDPA reversed micelles. In the new reversed micellar system, phase separation was very fast even at a high concentration of DTDPA.