Fructo-oligosaccharides (FOS) are a group of linear fructose oligomers with numerous health benefits, which can be synthesized by the transfructosylation of sucrose using fructosyltransferases (FTases). In this study, it was found that the selected FTases showed both hydrolytic and transfructosylating activities. For FOS production, the hydrolytic activities of FTases became significant when the reaction time was above 1 h. Thus, FTases needed to be inactivated or removed from the reactor after achieving the highest FOS conversion. We therefore developed an integrated ultrafiltration-diafiltration- concentration process for FTases reuse and FOS purification. Because of the 100% rejection of FTases activity and a higher average flux, a PES-10 ultrafiltration membrane was selected for FTases recovery, where the cake layer formation was the main membrane fouling mechanism. Then, a fed-batch reaction process was developed for FOS production, where the hydrolytic behavior of FTases was negligible, and the FTases dosage was reduced by 75% compared to single batch operation (four batches). Moreover, the FOS production was comparable, and 95.5% FTases was recovered by the PES10 membrane after the four fed-batches processes. Subsequently, a NF5 nanofiltration membrane was selected to further separate FOS from the FTases-free FOS solution by a constant volume diafiltration process under 45 °C, and the monosaccharides in the reaction mixture (glucose and fructose) were completely removed, increasing the FOS purity from 55.8% to 92.3%. Finally, the FOS and sucrose in the diafiltration permeate was recovered by a DL nanofiltration membrane under 60 °C. The outcome of this work offers a complete technical route for FOS production using sucrose as substrate.