Abstract: Nelumbo nucifera Gaertn. (sacred lotus) is not only an important aquatic vegetable crop, but also good material for basic scientific research. The extraction of sacred lotus seed proteins was optimized using four protein extraction methods (new TCA/acetone, traditional TCA/acetone, improved Tris-HCl, and Tris-saturated phenol methods) combined with two-dimensional electrophoresis (2-DE) technology. The 2-DE analysis showed that the resulting protein profile/map exhibited consistency with the composition and distribution of the sacred lotus seed proteome in ‘Tielian’. Analysis by PDQuest software showed that the new TCA/acetone method was suitable for 2-DE protein extraction of lotus cotyledons and plantules, whereas the traditional TCA/acetone method was suitable for the 2-DE protein extraction of hypocotyls. This work lays a foundation for further research on the sacred lotus seed proteome by mass spectrometry.