Cellulose binding domains (CBDs) are discrete protein modules found in most cellulolytic enzymes which are distinct from catalytic domains. The presence of a CBD improves the binding and facilitates the activity of the catalytic domain on the insoluble cellulose substrates. The objectives of this study were to synthesize cellulose binding domains for the later research on understanding the interaction between CBDs and cellulose and cellulose-based materials, as well as to generate CBD-integrated nanocomposites for cellulose fiber organization engineering. Fungal CBDs from Trichoderma reesei cellobiohydrolase 1 (CBH1) and cellobiohydrolase 2 (CBH2) were heterologously expressed in host strain Escherichia coli BL21 (DE3) by constructing CSD-pET31b expression recombinant. The CBD coding sequences were cloned downstream of a 125 amino acid ketosteroid isomerase (KSI) gene and upstream of a hexa-histidine tag sequence in pET31b(+) vector and cleaved by CNBr to release target CBD peptides. The expressed proteins were purified by Ni-NTA immobilized metal affinity chromatography (IMAC). The application of this expression approach represents a fast and efficient method to prepare cellulose binding domain in its biologically active form, which makes it possible to further create the cellulose binding domain-integrated molecular linker to engineer the organization of cellulose-base materials and to create new functional cellulose nanocomposites.