2'-Fucosyllactose (2'-FL) is the most abundant human milk oligosaccharide and plays significant roles in gut microbiome balance, neural development, and immunoregulation. However, current fermentation schemes using multiple carbon sources increase production cost and metabolism burden. This study reported the development of an engineered Bacillus subtilis strain that produces 2'-FL using glucose as the sole carbon source. First, a lactose biosynthesis module was constructed by expressing β-1,4-galactosyltransferase gene from Neisseria meningitidis. A 2'-FL titer of 2.53 ± 0.07 g/L was subsequently achieved using glucose as the sole carbon source by the combination of lactose and GDP-L-fucose (GDP-Fuc) biosynthesis modules. Introducing an exogenous nonphosphorylated transport system enhanced the supply of intracellular nonphosphorylated glucose, and the 2'-FL titer increased to 4.94 ± 0.35 g/L. Next, a transcription factor screening platform was designed. Based on this platform, the ligand of the transcription factor LacI was changed from isopropyl β-D-thiogalactoside to lactose. A lactose-responsive genetic circuit was then constructed and used for the dynamic regulation of metabolic fluxes between lactose and GDP-Fuc biosynthesis modules. Ultimately, the 2'-FL titer of the dynamically regulated strain improved by 107% to 9.67 ± 0.65 g/L in shake-flask, and the titer and yield in a 3-L bioreactor reached 30.1 g/L and 0.15 g/g using glucose as the sole carbon source. By using multidimensional engineering strategies, this study constructed a B. subtilis strain capable of efficiently producing 2'-FL with glucose as the sole carbon source, paving the way for the industrial production of 2'-FL with low cost in the future.