In this paper, the promotion effect of different biochar fillers on the electric generation performance of sedimentary microbial fuel cells (SMFCs) was studied. Three different biochar types were successfully prepared by high temperature pyrolysis and acidification modification technology. In order to evaluate their application value in improving the power generation efficiency of SMFCs, not only the specific surface area and weight loss rate of these biochar were measured. The output voltage, polarization curve and power density curve were also analyzed. The results showed that the specific surface areas of corn stalk, peanut shell and walnut shell were 24.66 m2/g, 3.90 m2/g and 3.67 m2/g, respectively. The SMFCs output voltages of the three biochar fillers were 0.16 V, 0.13 V and 0.19 V, respectively. The power density of SMFCs was higher than that of blank group (0.01 V). Moreover, the power density of SMFCs of corn stalk filler is as high as 91 mW/m3, which is nearly twice that of blank group (48 mW/m3). And the internal resistance of SMFC with corn stalk fillers (3981.63 Ω) is also lower than that of the other groups. It can be seen that biochar fillers can further optimize the power generation performance of sedimentary microbial fuel cells, and provide a new choice for the development direction of low-cost, green and clean SMFC.