Chitin binding lectin, found in seeds of Datura stramonium, (DSL), is an important glycan binding protein that has great therapeutic properties. The objective of the study is to understand the evolutionary significance, structural and functional characterization of chitin binding lectin from D. stramonium, thus will facilitate to explore in deeper structural insights about the protein and its interactions with substrates. In this study, initially the sequence analysis was performed for chitin binding lectin to understand the sequential properties followed by using similarity search, multiple sequence alignment and phylogenetic analysis to identify the closely related protein sequences of DSL. After this, we utilized hybrid homology modeling-ab initio approaches to predict the 3D model of DSL, which is subsequently used for interaction studies with four ligands namely N,N'-Diacetylchitobiose, Triacetylchitotriose and Chitin tetramer, which are all oligomers of chitin. Docking analysis was also performed for N-Acetyllactosamine, which is reported as a potent inhibitor of haemagglutination by Datura lectin.Interestingly we observed two binding sites of substrate. The active site residues in predicted binding site are Glu272, Arg62 and Thr246. Moreover, the best four DSL-ligand complexes along with unbounded form of DSL were subjected to MD simulation to understand the structural stability, integrity and compactness. Together the results of docking and MD simulation, the chitotriose oriented in center of the DSL showing more binding affinity towards binding pocket of DSL. This comprehensive analysis of DSL provides key insights about the structure, active site, binding affinity and mode of binding of the substrates.