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 µM. 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.