In human, acetylcholinesterase (AChE) is a cholinergic enzyme involved in the hydrolysis of neurotransmitter acetylcholine (ACh) into its constituents, choline, and acetate. In plants, the biological functions of AChE are lacking and its existence has been recognized by indirect evidence of its activity. Therefore, in the present investigation, a systematic analysis of the AChE gene family in tomato was performed by integrating structural features, phylogenetic analysis, and its enzyme activity. Using the computational approach, we have identified 87 SlAChE genes containing GDSL lipase/acylhydrolase domain in tomato. In silico expression analysis of SlAChE genes showed up-and down regulation under salinity stress condition. The activity of the AChE enzyme was further confirmed using Ellman assay. Promoter analysis of SlAChE genes using PlantCARE showed the presence of several cis-acting elements including abiotic stress, light, and hormone regulatory elements. In silico screening indicated that tomato AChE homologs are widely distributed in plants. Syntenic analysis revealed several gene pairs between tomato and other species. Interestingly, the deduced amino acid sequence of human AChE showed no similarity with that of tomato AChE sequence. However, the binding energy of SlAChE enzyme to agonists and antagonists was almost identical to that of human AChE. This preliminary study of ChE-like activity in plants may open the way for additional research in non-neuronal role in plants. The studies provide a theoretical basis for further elucidating the functions of the AChE gene family at the molecular level.