This work describes the reuteransucrase-catalyzed reaction and structural characterization as well as in vitro fermentation for the acceptor products of gluco-oligomers from sucrose and maltose. At a low concentration of sucrose, the production of gluco-oligomers was favored, resulting in a relatively large number of acceptor products (DP_3–5). A mathematical model was also proposed to simulate gluco-oligomer production depending on the reaction conditions. The fine structures of major linear gluco-oligomer fractions for a sucrose : maltose ratio of 1 : 1 were assigned as follows: α-D-Glcp-(1→6)-α-D-Glcp-(1→4)-D-Glcp, α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-D-Glcp, α-D-Glcp-(1→4)-α-D-Glcp-(1→6)-α-D-Glcp-(1→4)-D-Glcp, and α-D-Glcp-(1→6)-α-D-Glcp-(1→4)-α-D-Glcp-(1→6)-α-D-Glcp-(1→4)-D-Glcp, respectively. Compared with dextran and GOS57, the results of fermentation selectivity indicated that gluco-oligomers promoted the proliferation of gut bacteria and total SCFA production with a higher concentration of propionate. These data suggested that the gluco-oligomers synthesized via the reuteransucrase acceptor reaction had a prebiotic effect on gastrointestinal health.