Abstract Panose is a mildly sweet trisaccharide composed of three glucose units which has the nature of an anti-cariogenic sugar in foods. Continuous production of panose from pullulan was investigated by using a column of immobilized neopullulanase. Although enzyme immobilization by ionic bond is one of the most general methods for the production of non-ionic materials from non-ionic substrates, the activity of the neopullulanase immobilized on DEAE-cellulose significantly decreased. When the neopullulanase was immobilized on a carrier with spacer arms by covalent bond through the formation of the schiff base, the activity was fully expressed. Optimum temperature and pH for the reaction of the immobilized neopullulanase were 55–60°C and 6.0, which were almost the same as those of the free enzyme. The immobilized neopullulanase column was employed for the continuous production of panose from pullulan. More than 92% of pullulan was converted by this system into the final products; panose, maltose, and glucose. The ratio of panose in the mixture of products was 84%, which was significantly higher than that (around 70%) of a batch system using the free enzyme. The immobilized neopullulanase was very stable, and more than 90% of the initial activity was retained after 150-h continuous reaction at 55°C.