Carbonaceous materials derived from a variety of cheap and sustainable biomass have shown great potential as conductive substrates for the coating of pseudocapacitive materials. In this study, yeast-derived N-doped carbon microsphere/polyaniline (YC/PANI) composites are synthesized by an in situ polymerization approach, using yeasts as the carbon source and structural template. The self-templated hollow YC with good conductivity has many active sites for anchoring PANI, and alleviates the structure destruction caused by the redox reaction of PANI. Accordingly, the YC/polyaniline composite as a supercapacitor electrode exhibits a high specific capacitance (500 F g−1 at a current density of 1 A g−1) and good rate capability in a three-electrode system. Additionally, the symmetric supercapacitor of the YC/polyaniline composite delivers a high capacitance (100 F g−1 at 1 A g−1) and remarkable cycle life (95.4% of the initial capacitance maintenance after 5000 cycles). The uniform coating of PANI nanowires and robust structure stability of composites are responsible for excellent electrochemical properties.