Spodoptera litura is a threatening agricultural insect in tropical and subtropical areas and accounts for tremendous annual crop losses. As seen in virtually all insect species, serine proteases (SPs) are crucial to S. litura. The expression pattern of SPs from the midgut of S. litura was studied through expressed sequence tags (ESTs) analysis. One of SP (SlSP1) was chosen for detailed study, because the expression of the gene was midgut and larvae specific. SlSP1 was conducted as a model of its evolution, structure, and potential binding activity with corresponding substrates. SlSP1 is composed of 255 amino acids including a signal peptide at N-terminal followed by a putative activation peptide and the mature protein along with five putative phosphorylation sites, three disulphide bridges, and two N-glycosylation positions. At least nine conserved motifs were obtained in multiple sequence alignments. Some conserved residues, such as the catalytic triad His84, Asp127, and Ser229 as well as six cysteines at position 66, 82, 194, 211, 223, and 247, were examined. After homology modeling and molecular dynamics simulation, the resultant three-dimensional (3D) structure of SlSP1 was docked with the substrates 2PTC-Arg and 2PTC-Lys, respectively. Molecular Mechanic/Poisson–Boltzmann surface area analysis was applied to anticipate optimal binding mode and crucial active sites of this enzyme. The residues Trp28, Gly187, Aso188, Arg249, Ile250, Lys246, and Lys278 are crucial for the substrate binding and molecule process. This information can be used in logical design of SPs inhibitors. New inhibitors may be a basis for development of a new pest control technology.