Polyacrylonitrile (PAN) nanofibers, with and without embedded carbon nanotubes (CNTs) were fabricated by the electrospinning process. Polypyrrole (PPy) was coated on the activated PAN/CNT nanofiber by in-situ chemical polymerization in order to improve the electrochemical performance. The electrocatalytic behaviors of the PPy-PAN/CNT composite anode were investigated by means of cyclic voltammetry to evaluate as the anode for microbial fuel cells (MFCs) application. In comparison with unmodified carbon cloth (CC) anodes, PPy-PAN/CNT nanofiber composite showed the improvement of the maximum power density by 40%. The PPy-PAN/CNT nanofiber composite electrode therefore offers good prospects for application in MFCs.