The precipitation phase boundary of trisodium salt of 2-(2-carboxyethyl)-3-decyl maleic anhydride (DCMA-3Na) was determined with various metal ions, such as Ca+2, Mg+2, Zn+2 Cu+2, and Al+3. The tricarboxylic-acid-type biosurfactant DCMA-3Na was synthesized from spiculisporic acid. Solid precipitates were analyzed by FT-IR and an optical microscope. The precipitation boundary diagrams were prepared through both the measurement of the supernatant solution concentrations and the visual inspection of precipitate formation. The multivalent ion concentration necessary to cause the precipitation of the surfactant (i.e., tolerance) decreased as the DCMA-3Na concentration increased, up to a certain critical concentration in the same way as with chemically synthesized commercial surfactants such as SDS. However, unlike the commercial surfactants, the tolerance of DCMA-3Na toward hardness ions did not increase when the concentration of surfactant increased further above the critical point. This interesting phenomenon is due to the fact that excess amounts of sodium ions dissociated from the three carboxylate groups in the head group of DCMA-3Na compete with the multivalent ions over binding onto the micelle surface.