Ajwain (Trachyspermum ammi) belongs to the family Umbelliferae, is commonly used in traditional, and folk medicine due to its carminative, stimulant, antiseptic, diuretic, antihypertensive, and hepatoprotective activities. Non-specific lipid transfer proteins (nsLTPs) reported from various plants are known to be involved in transferring lipids between membranes and in plants defense response. Here, we describe the complete primary structure of a monomeric non-specific lipid transfer protein 1 (nsLTP1), with molecular weight of 9.66 kDa, from ajwain seeds. The nsLTP1 has been purified by combination of chromatographic techniques, and further characterized by mass spectrometry, and Edman degradation. The ajwain nsLTP1 comprises of 91 amino acids, with eight conserved cysteine residues. The amino acid sequence based predicted 3D structure is composed of four α-helices stabilized by four disulfide bonds, and a C-terminal tail. The predicted model was verified by using different computational tools; i.e. ERRAT, verify 3D web server, and PROCHECK. The docking of ajwain nsLTP1 with ligands; myristic acid (MYR), and oleic acid (OLE) was performed, and MD (Molecular Dynamics) simulation was used to validate the docking results. The findings suggested that amino acids; Leu11, Leu12, Ala55, Ala 56, Val15, Tyr59, and Leu62 are pivotal for the binding of lipid molecules with ajwain nsLTP1.