Abstract: To improve the yield of D-tagatose, the initial step of this study was to screen and identify microorganisms capable of efficiently producing L-arabinose isomerase (L-AI) from soil samples. Subsequently, the araA gene was cloned and heterologously expressed to obtain L-AI. To enhance the synthesis efficiency of D-tagatose, L-AI and β-galactosidase (β-GAL) were co-immobilized on the prepared PVA/CNTs/ZnO carrier to construct a dual-enzyme reaction system. In addition, PVA/CNTs/ZnO immobilization carrier was analyzed by scanning electron microscopy (SEM), thermogravimetric (TG), Fourier transform infrared spectroscopy (FT-IR), and X-ray photoelectron spectroscopy (XPS). Furthermore, the optimal reaction temperature and pH of dual-enzyme cascade by the PVA/CNTs/ZnO@L-AI/β-GAL were investigated. In this study, a high-yield D-tagatose-producing strain named LW48, was identified as Bacillus amyloliquefaciens. The recombinant L-AI protein was obtained by cloning and expression. The dual-enzyme cascade of β-GAL and L-AI immobilized on the PVA/CNTs/ZnO carrier, along with the carrier itself, was characterized by SEM, TG, FT-IR, and XPS. The results showed that the PVA/CNTs/ZnO carrier had excellent stability. When lactose was used as the substrate, the optimal temperature and pH for L-AI were 65 ℃ and pH 7.0, respectively. The kinetic parameters for the v_max and K_m of dual-enzyme cascade (β-GAL and L-AI) were 0.187 mg·L^-1·s^-1 and 6.02 mg·L^-1, respectively. The v_max and K_m of the free enzymes (β-GAL and L-AI) were 0.230 mg·L^-1·s^-1 and 22.95 mg·L^-1, respectively. After 24 h of reaction under optimal conditions, the maximum conversion rate of the free enzymes was 13.2%. The maximum conversion rate of immobilized cascaded dual enzymes (L-AI and β-GAL) reached 42.9%, which was 3.25 times that of free enzymes(L-Al and β-GAL). In this study, the preparation of PVA/CNTs/ZnO carrier effectively improved the biosynthesis efficiency of β-GAL and L-AI dual-enzymes cascade for the green production of D-tagatose, providing a new approach for the industrialization of D-tagatose, with the efficient and green bio-manufacturing of rare sugars.