A bienzyme biosensor in which the enzymes β-galactosidase (β-Gal), fructose dehydrogenase (FDH), and the mediator tetrathiafulvalene (TTF) were coimmobilized by cross-linking with glutaraldehyde atop a 3-mercaptopropionic acid (MPA) self-assembled monolayer on a gold disk electrode, is reported. The working conditions selected were Eapp=+0.10 V and (25±1) °C. The useful lifetime of one single TTF-β-Gal-FDH-MPA-AuE was surprisingly long, 81 days. A linear calibration plot was obtained for lactulose over the 3.0×10−5–1.0×10−3 mol L−1 concentration range, with a limit of detection of 9.6×10−6 mol L−1. The effect of potential interferents (lactose, glucose, galactose, sucrose, and ascorbic acid) on the biosensor response was evaluated. The behavior of the SAM-based biosensor in flow-injection systems in connection with amperometric detection was tested. The analytical usefulness of the biosensor was evaluated by determining lactulose in a pharmaceutical preparation containing a high lactulose concentration, and in different types of milk. Finally, the analytical characteristics of the TTF-β-Gal-FDH-MPA-AuE are critically compared with those reported for other recent enzymatic determinations of lactulose.