Bradyrhizobium japonicum (BJ), which has a symbiotic relationship with soybean roots, possessed an abundant three-dimensional (3D) structure with a high N content. A soybean leaf (SL) with a hierarchically ordered macroporous network and numerous polar hydroxyl groups was proposed as a separator for a supercapacitor. A 3D hierarchical porous carbon was prepared by the facile carbonization with chemical activation of BJ. The as-prepared material possesses a large specific surface area (1275 m2·g–1), unique 3D hierarchical porosity, and good electrical conductivity. The electrochemical performance of the Bradyrhizobium japonicum-derived porous carbons at a mass ratio of 1.5 (ZnCl2/BJ = 1.5) (BJPC-1.5) for supercapacitors with a SL separator showed a high capacitance (358 F/g at 1 A·g–1), a superior cycle stability of 91% over 8000 cycles, and a superior rate capability in a symmetric two-electrode supercapacitor in 6 M KOH. Furthermore, the electrochemical performance of the BJPC-1.5 with a SL separator was comparable to that with the commercialized cellulose and polypropylene (PP) separators. More attractively, the SL separator with a preferable water uptake showed a much better performance in the BJPC-1.5 cell than the PP separator. These results provide an insight into the full usage of a natural and biodegradable biomass for separator and electrode materials for a supercapacitor.