In this study, we investigated whether galactooligosaccharide (GOS) can be stably and steadily synthesized using immobilized β-galactosidase (β-gal) inclusion body (IB)- containing E. coli cells during long-term repeated-batch operation. To improve the operational stability of this enzyme reactor system, immobilized E. coli cells were crosslinked with glutaraldehyde (GA) after immobilization of the E. coli. When we treated with 2% GA for E. coli crosslinking, GOS production continued to an elapsed time of 576 h, in which seven batch runs were operated consecutively. GOS production ranged from 51.6 to 78.5 g/l (71.2 ± 10.5 g/l, n=7) during those batch operations. In contrast, when we crosslinked E. coli with 4% GA, GOS production ranged from 31.5 to 64.0 g/l (52.3 ± 10.8, n=4), and only four consecutive batch runs were operated. Although we did not use an industrial β-gal for GOS production, in which a thermophile is used routinely, this represents the longest operation time for GOS production using E. coli β-gal. Improved stability and durability of the cell immobilization system were achieved using the crosslinking protocol. This strategy could be directly applied to other microbial enzyme reactor systems using cell immobilization to extend the operation time and/or improve the reactor system stability.