This study presents the identification and characterization of the promoter region of Vibrio anguillarum NB10, which enhances the expression of FK506-binding protein (FKBP)-type peptidyl-prolyl cis/trans isomerase (PPIase; FklB), capable of binding to the immunosuppressant FK506 under extremely alkaline conditions. Our proteomic analysis of V. anguillarum NB10 revealed that FklB (VaFklB) expression is significantly upregulated under extreme alkaline stress (pH 10). When the putative core promoter regions were coupled with a β-galactosidase reporter gene and introduced into Escherichia coli, we observed β-galactosidase activities of 61.47 ± 2.91 and 95.83 ± 6.76 Miller units (MU) at pH 9 and 10, respectively, after 4 h of stress exposure. These values represent 1.97- and 2.88-fold increases compared to normal conditions (25 °C, pH 7: 31.27 ± 1.15 MU). This alkaline-inducible promoter system has potential for biotechnological applications, including the development of pH-responsive gene expression systems, biomanufacturing processes requiring alkaline environments, and targeted activation of silent biosynthetic gene clusters for novel bioactive compound discovery. Our findings provide a valuable molecular tool for synthetic biology and metabolic engineering, enabling precise genetic control under specific environmental conditions that may revolutionize industrial biotechnology.