Multienzyme systems are essential for utilizing di-, oligo-, and polysaccharides as fuels in enzymatic fuel cells effectively. However, the transfer of electrons generated by one enzymatic reaction in a multienzyme cascade at the electrode may be impeded by other enzymes, potentially hindering the overall efficiency. In this study, carbon paper was first modified by incorporating single-walled carbon nanotubes (SWCNTs) and gold nanoparticles (AuNPs) sequentially. Subsequently, glucose oxidase (GOx) and a trehalase-gelatin mixture were immobilized separately on the nanostructured carbon paper layer-by-layer adsorption to mitigate the electron transfer hindrance caused by trehalase. The anode was first fabricated by immobilizing GOx and trehalase on the modified carbon paper, and the cathode was then fabricated by immobilizing bilirubin oxidase on the nanostructured electrode. The SWCNTs and AuNPs were distributed adequately on the electrode surface, which improved the electrode performance, as demonstrated by electrochemical and morphological analyses. An enzymatic fuel cell was assembled and tested using trehalose as the fuel, and a maximum power density of 23 μW cm was obtained at a discharge current density of 60 μA cm. The anode exhibited remarkable reusability and stability.