Both of the topographical and gradient conductive cues can influence the cellular activity and thereby tissue regeneration. However, they have not been combined simultaneously onto biomaterial with electrical stimulation to demonstrate the synergistic role so far. Herein, we assume that a bacterial cellulose (BC) -based membrane by incorporating aligned nanofibers and a concentration gradient of polypyrrole (PPy) with electrical stimulation treatment will promote cell differentiation in peripheral nerve regeneration. The results showed that PPy were successfully deposited on the aligned BC/PPy with gradient conductive structure, which exhibited good mechanical property, thermal stability, the gradient decrease in surface resistance, gradient increase in surface current from the up to down segments, as well as excellent biocompatibility. Especially, the membranes promoted the gradient proliferation and differentiation of PC12 cells in vitro. Importantly, combined with electric field (EF), the aligned BC/PPy gradient conductive membranes synergistically directed the differentiation of PC12 cells. The overall results suggest the aligned BC/PPy gradient conductive membranes with EF could be a promising therapeutic strategy to guide cellular activities for peripheral nerve regeneration.