Bacteria cellulose (BC) nanofibers are used as robust biotemplates for the facile fabrication of novel gold nanoparticle (NP)-bacteria cellulose nanofiber (Au-BC) nanocomposites via a one-step method. The BC nanofibers are uniformly coated with Au NPs in aqueous suspension using poly(ethyleneimine) (PEI) as the reducing and linking agent. With the addition of different halides, Au-BC nanocomposites with different Au shell thicknesses are formed, and a possible formation mechanism is proposed by taking into account the special role played by PEI. A novel H2O2 biosensor is constructed using the obtained Au BC nanocomposites as excellent support for horseradish peroxidase (HRP) immobilization, which allows the detection of H2O2 with a detection limit lower than 1 mu M. The Au-BC nanocomposites could be further used for the immobilization of many other enzymes, and thus, may find potential applications in bioelectroanalysis and bioelectrocatalysis.