In order to search for high activity acetylcholinesterase (AChE) inhibitors, thirteen 2H-thiazole[3,2-b][1,2,4]triazine-3,7-diones were synthesized from substituted benzaldehyde and N-Acetylglycine through five steps. The structures of the target compounds were confirmed by 1H NMR, 13C NMR and HRMS. Ellman method was used to test AChE inhibitory activity of the compounds at 10 μmol/L. The results showed that all the target compounds had inhibitory activity against AChE, and the compound 6-(4-methoxybenzyl)-2-(4-methoxybenzylidene)-2H-thiazolo[3,2-b]-1,2,4-triazine-3,7-dione (Ⅴk) had the best activity (80.1%). The binding mode of the compound Ⅴk and AChE was studied by molecular docking. The compound Ⅴk could bind to multiple catalysis sites of AChE. 2H-thiazole[3,2-b][1,2,4]triazine-3,7-diones could provide reference for the development of new AChE inhibitors with independent intellectual property rights.