Based on the "cluster effect" and the structure characters of acetylcholinesterase (AChE; EC 3.1.1.7), a new series of 1,2,4-triazolin-3-one and phthalimide heterodimers were designed, synthesized, and evaluated as potent dual acetylcholinesterase inhibitors (AChEIs). Most of the synthesized compounds showed good in vitro inhibitory activities towards both Drosophila melanogaster acetylcholinesterase (DmAChE) and Musca domestica acetylcholinesterase (MdAChE). Among them, 5g was found to be the most potent anti-AChE derivate (5g, IC = 8.07 µM to DmAChE, IC = 32.24 µM to MdAChE). It was 2.31- and 1.35-fold more active than the positive control ethion (CP, IC = 18.62 µM to DmAChE, IC = 43.56 µM to MdAChE). The docking model study revealed that 5g possessed the fitted spatial structure and bound to the central pocket and peripheral site of DmAChE. Moreover, most compounds demonstrated high insecticidal activity to Lipaphis erysimi and Tetranychus cinnabarinus at the concentration of 300 mg/L.