Bi- and Cu-substituted Ca3Co4O9 samples were prepared by conventional solid-state reaction method and the effect of element substitution on the microstructures and thermoelectric properties was investigated. Partial substitution of Cu for Co leads to an increase in electrical conductivity and a decrease in Seebeck coeffcient due to the rise of hole concentration. The microstructure of Cu-substituted sample is almost unchanged compared with undoped Ca3Co4O9. On the other hand, partial substitution of Bi for Ca gives rise to a significant increase in the grain size, and c-axis-oriented structure can be formed in Ca2.7Bi0.3Co4O9, resulting in an obvious increase in electrical conductivity. Cu and Bi co-substitution further increases the grain growth and the electrical conductivity of Ca2.7Bi0.3Co3.7Cu0.3O9. Thus, Cu and Bi co-substitution samples possess the optimal thermoelectric performance at high temperature and the highest value of power factor can reach 3.1×10-4 Wm-1·K-2 at 1000 K. Bi- and Cu-substituted Ca3Co4O9 samples were prepared by conventional solid-state reaction method and the effect of element substitution on the microstructures and thermoelectric properties was investigated. Partial substitution of Cu for Co leads to an increase in electrical conductivity and a decrease in Seebeck coeffcient due to the rise of hole concentration. The microstructure of Cu-substituted sample is almost unchanged compared with undoped Ca3Co4O9. On the other hand, partial substitution of Bi for Ca gives rise to a significant increase in the grain size, and c-axis -oriented structure can be formed in Ca2.7Bi0.3Co4O9, resulting in an obvious increase in electrical conductivity. Cu and Bi co-substitution further increases the grain growth and the electrical conductivity of Ca2.7Bi0.3Co3.7Cu0.3O9. Thus, Cu and Bi co-substitution samples possess the optimal thermoelectric performance at high temperature and the highest value of power factor can reach 3.1 × 10-4 Wm-1 · K-2 at 1000 K .