In the present study, a new strain of Lactobacillus brevis producing D-tagatose was isolated and identified. Then, the L-arabinose isomerase (L-AI) of this strain was displayed on the spore surface of Bacillus subtilis DB403 by using an anchoring protein CotG and a peptide linker (Gly-Gly-Gly-Gly-Ser). This displayed L-AI with high specific activity and stability was used as a novel immobilized biocatalyst for producing D-tagatose through batch and semi-continuous biotransformation. The conversion rate of D-tagatose from 125 g/L D-galactose was achieved 79.7% at 28 h, and the volumetric productivity reached 4.3 g/L/h at 20 h. Furthermore, the displayed L-AI showed a good performance on the reusability and remained 87% of the specific activity and 40.7% of the conversion rate after five recycles. A high efficient immobilized method for producing food-grade D-tagatose was established using spore surface-displayed L-AI.