An organic solvent-tolerant β-fructofuranosidase (β-FFase) from Arthrobacter arilaitensis NJEM01 was purified, characterized, cloned, and overexpressed in Escherichia coli. The mature β-FFase contained 495 amino acid residues with an estimated molecular mass of 55 kDa. The purified β-FFase from strain NJEM01 was very stable in the buffer systems (pH 5.0-9.5) and showed high stability below 45 °C. Furthermore, the enzyme exhibited relatively high solvent stability in various aqueous organic mixtures and retained nearly 100% of its initial activity after incubation for 10 days in 20% (v/v) DMSO. In addition, the β-FFase exhibited high transfructosylation activity, synthesized prebiotic products of mainly 6-kestose (up to 476 g/L), and showed fructosyl receptor specificity to C-glucosyl flavone. A relatively high yield of FOS was achieved by the β-FFase from bacterium with a high concentration of sucrose. It made the β-FFase an exploitable biocatalyst for the production of glycosides of natural products and prebiotic kestose.