Cellobiose is one of the most abundant carbohydrates available in nature which can be used as biofuel in various applications such as food industry, energy production, paper production, etc. with no harm to the environment. One way for a safe decomposition the cellobiose is enzymatic hydrolysis. Cellobiohydrolase enzyme, which is naturally present in some yeasts and bacteria, is capable of decomposing carbohydrates. Because of its low production in its original organism and also high volumes of requirements to this enzyme for industrial usage, there is strong desired to mass-produce of this enzyme using biotechnological approaches. In this study, several signal peptide sequences with Lactococcus originality, with the aim of binding to the cellobiohydrolase gene, were evaluated. In this regard, signalP server, a valid and accurate online tool, was used to determine the most important details including N-terminal (n), hydrophobic (h) and carboxy-terminal (c) regions of signal peptides and their probability. Thereafter, Portparam and Solpro online tools were used to investigate the physicochemical properties of each signal peptide. Finally, the best peptide signal with the best specifcity and the highest D-score was selected for binding to the cellobiohydrolase gene sequence aimed at expression in the Gram-positive bacterium Lactococcus lactis. Final frame of target gen was synthetized and ligated in pBU003 expression vector using double digestion by SalI and XhoI restriction enzymes. Recombinant expression vector was transferred to MC1061 for replication and then transferred into the MG1363 as expressing strain of Lacticoccus lactis. Recombinant proteins were purifed from M17 medium using the Ni-NTA Agarose column. Their quantifcation and qualifcation examined through Bradford assay and 12% SDS-PAGE respectively. Phosphoric acid-treated cotton as a substrate was used to determine the Ce16B cellobiohydrolase activity at 450 nm absorbance. In silico investigating the signal peptides have led to the choice of the USP45 signal peptide as a powerful secretory signal. Recombinant proteins were successfully expressed in Lactococcus lactis. The concentration of each recombinant protein was determined 0.7 g/l (R2 = 0.989). In vitro assessment of enzymatic activity or recombinant proteins on PC showed 1.89 U (R2 : 9941).