Abstract: The invention provides a bacterial cellulose-based biosensor, including bacterial cellulose (BC) and a cell presenting a cellulose-binding module CBM2a on the surface. The cell is attached to BC through CBM2a. The cell expresses CBM2a by using pETDuet-tac as a vector. The pETDuet-tac is obtained by replacing two T7 promoters on the vector pETDuet by tac promoters, that is, an upstream first tac promoter and a downstream second tac promoter. The pETDuet-tac includes a gene encoding a fluorescent protein downstream of the first tac promoter and a gene encoding CBM2a presented on the surface downstream of the second tac promoter. By the BC-based biosensor, efficient and specific immobilization of cells on the BC matrix is enabled, the biological activity of cells is maintained, the fluorescence signal output is enhanced, and sufficient pores are provided for the entry and exit of a detected substance, thereby significantly improving the detection sensitivity.