Bacillus subtilis strain AP-1, which produces α-glucosidase with transglucosidase activity, was used to produce a series of long-chain isomaltooligosaccharides (IMOs) with degree of polymerization (DP) ranging from 2 to 14 by direct fermentation of maltose. A total IMOs yield of 36.33 g/L without contabacillusmination from glucose and maltose was achieved at 36 h of cultivation using 50 g/L of maltose, with a yield of 72.7%. IMOs were purified by size exclusion chromatography with a Superdex 30 Increase column. The molecular mass and DP of IMOs were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). Subsequently, linkages in produced oligosaccharides were verified by enzymatic hydrolysis with α-amylase and oligo-α-1,6-glucosidase. These IMOs showed prebiotic properties, namely tolerance to acidic conditions and digestive enzymes of the gastrointestinal tract, stimulation of probiotic bacteria growth to produce short-chain fatty acids and no stimulating effect on pathogenic bacteria growth. Moreover, these IMOs were not toxic to mammalian cells at up to 5 mg/mL, indicating their biocompatibility. Therefore, this research demonstrated a simple and economical method for producing IMOs with DP2–14 without additional operations; moreover, the excellent prebiotic properties of the IMOs offer great prospects for their application in functional foods.