SummaryIsomalto‐oligosaccharides (IMO) belong to a group of prebiotics that can significantly increase the number of protective gut microflora. A one‐step method using neopullulanase (NPN) in conjunction with saccharifying α‐amylase (SAA) for the bioconversion of rice starch into IMO was investigated. Purified rice starch slurry (30% w/w) was mixed with NPN (3.5 U g−1 starch substrate) and SAA (6.5 U g−1 starch substrate) and the slurry was incubated at 57 °C for 92 h under constant stirring. The carbohydrate composition of the resulting syrup was analysed by high performance liquid chromatography (HPLC) and the dextrose equivalent (DE) determined by titration. The amount of IMO in the syrup reached maximum (59.2%, dry basis) after 72 h of bioconversion. The concentration of glucose and maltose, which were the main carbohydrate residues of the IMO syrup, was 25.5% while the concentration of other oligosaccharide residues was about 1.0%. The results demonstrate that rice starch is a suitable matrix for producing IMO syrup and the one‐step conversion procedure appears to be an efficient method for converting starch into IMO syrup.