In this study, mesoporous polyacrylonitrile (PAN)/thermoplastic polyurethane (TPU) blended nanofibers were prepared to immobilize Micrococcus luteus for enhancing the conversion of trehalose. The images of SEM showed the cells were adsorbed on the surface and pores due to the unique pore structure. The results of contact angle, Zeta potential and water holding ratio exhibited the good hydrophilicity and stability of PAN/TPU-P2. Besides, it was indicated that the biomass and immobilization efficiency were increased to 0.633 g/L and 0.153 g/g, respectively. It was the most noteworthy that the trehalose yield could reach 23.46 g/L, which was 71.62 % higher than that of the control in the multi-batch fermentation. Moreover, the reactive oxygen species (ROS) level was decreased to 12.8 % while the enzyme concentration was increased to 11.176 mg/mL. Meanwhile, it was also found that PAN/TPU-P2 immobilization substantially increased the expression of target gene MtreY by 3.500 times. In other words, the mechanism by which immobilized cells increased trehalose yield was that PAN/TPU-P regulated gene expression of MtreY. Therefore, this research provided theoretical foundation for the metabolic regulation of sufficient trehalose production by immobilized cells.