This study developed a simple two-step procedure to produce isomaltooligosaccharides (IMOs) from low-cost sweet potato starch (SPS). Effect of various reaction parameters on the steps of the synthesis process of IMOs was systematically investigated. The results show that Spezyme Xtra enzyme was the most suitable for the liquefaction step. The oligosaccharide components’ contents, including G1–G10 and G2–G6, reached 73.95 ± 0.02% and 49.24 ± 3.19%, respectively, after liquefaction. The simultaneous saccharification and transglycosylation (SST) reaction of SPS followed the liquefaction after the α-amylase activity was deactivated. This reaction was simultaneously treated by β-amylase, pullulanase, and α-transglucosidase. The effect of various reaction parameters, consisting of solution pH, reaction temperature, enzyme dosage, and reaction time, on the SST reaction to synthesize IMOs from SPS was fully studied. The results showed that the highest concentration of IMOs (IG234) reached 68.85 ± 1.82 g/L at the optimal condition. The purification of pristine IMO was performed by adding Saccharomyces cerevisiae var. diastaticus BE 134 yeast cells at the final step of the procedure. In particular, the SST reaction for the synthesis of IMOs from SPS shortened SST reaction time by three times compared with other three-step synthesis procedures of IMOs. These findings show that the SPS-derived IMOs can be applied as a novel and inexpensive prebiotic healthcare product for human gastrointestinal health, dieters, and diabetics.