Abstract: To improve the yield of D-tagatose, in this study, from soil samples, the initial step was to screen and identify microorganisms capable of efficiently producing L-arabinose isomerase (L-AI). Subsequently, the gene of araA 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 with the prepared PVA/CNTs/ZnO carrier to construct the multi-enzyme reaction system. In addition, PVA/CNTs/ZnO immobilization carrier was analyzed by scanning electron microscopy (SEM), thermogravimetric (TG), infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). Furthermore, the temperature and pH of PVA/CNTs/ZnO@L-AI@β-GAL were investigated. In this study, the high-yielding D-tagatose strain named LW 48, which was identified as Bacillus amyloliquefaciens. The recombinant protein L-AI was obtained by cloning and expression. Dual-enzyme cascade of β-GAL and L-AI immobilized on the PVA/CNTs/ZnO carrier was expressed and analyzed by SEM, TG, FTIR and XPS characterization. The results showed that the PVA/CNTs/ZnO carrier had excellent stability. When lactose was used as substrate, the optimal temperature and pH of L-AI were 65 ℃ and pH 7.0, respectively. The kinetic parameters on the v_max of dual-enzymes cascade (β-GAL and L-AI) was 0.187 g·L^-1·s^-1, and the K_m was 6.02 g·L^-1. The kinetic parameters on the v_max of the free enzymes (β-GAL and L-AI) was 0.230 g·L^-1·s^-1 and the K_m was 22.95 g·L^-1. After 24 h of the reaction under the optimal conditions, the maximum conversion rate of the free enzymes was 13.2%, and the maximum conversion rate of dual-enzymes cascade was 42.9%. The conversion rate of the latter was significantly higher (about 3 times) than that of the former. In our research, with the preparation of PVA/CNTs/ZnO carrier, the biosynthesis level of dual-enzymes cascade of β-GAL and L-AI for green production of D-tagatose was effectively improved, providing a new approach for industrial of D-tagatose and rare sugars with the efficient green bio-manufacturing.