Display omitted •A novel layer-by-layer in situ culture (LBLC) method was developed.•Bacterial cellulose/graphene/polyaniline nanocomposites were made by LBLC and polymerization.•The as-prepared nanocomposites are mechanically strong and highly flexible.•The as-prepared nanocomposites show excellent gravimetric capacitance and cycling stability. Rational structure, mechanical robustness, high conductivity, and favorable flexibility are important requirements for superior electrodes, which should not only possess high capacitance but also have freestanding structure without collector to improve the overall performance of supercapacitors. Herein, we demonstrate the fabrication of three-dimensional (3D) porous graphene-containing nanocomposites with highly dispersed graphene (GE) nanosheets in a 3D matrix of bacterial cellulose (BC) by a novel layer-by-layer in situ culture (LBLC) method. The BC/GE nanocomposites are then deposited with polyaniline (PANI), leading to the formation of BC/GE/PANI nanocomposites. Mechanical tests demonstrate excellent robustness and flexibility of the as-prepared BC/GE/PANI nanocomposites, which are used as electrodes directly without any nickel foam or stainless steel wire. The BC/GE/PANI electrode with an optimal GE content has a specific capacitance of 645 F g−1 at a current density of 1 A g−1, which is 2.5 times higher than that of BC/PANI and superior to most previously reported PANI-based electrodes. In addition, the symmetric supercapacitor assembled with BC/GE/PANI demonstrates a high energy density of 14.2 Wh kg−1 at a power density of 200 W kg−1. The excellent electrochemical performance of this BC/GE/PANI electrode is due to its unique 3D porous structure with the uniform distribution of GE nanosheets in the BC matrix and even PANI on BC nanofibers and GE nanosheets, which makes it very promising for diverse flexible energy storage devices. The methodology presented in this work can be extended to the preparation of other BC-based nanocomposite electrodes.