Cellobiose 2-epimerase (CE) plays a crucial role in catalyzing the conversion of lactose. In this study, the N-terminal 20 amino acids of Lactobacillus amylovorus feruloyl esterase (N20) were employed as a signal peptide and fused with the CE gene from Caldicellulosiruptor bescii for recombinant expression. Following ligation with the pET-22b(+) vector, Escherichia coli BL21 (DE3) was transformed. SDS-PAGE analysis confirmed the extracellular secretion of the CE following fusion with the signal peptide. Following fermentation optimization to maximize extracellular protein secretion, the optimal conditions were identified as a 2 × YT medium, supplemented with 0.8 mM IPTG, 0.1 mM ferrous ion (Fe