Staphylococcus aureus (S. aureus) is one of the prominent gram positive human pathogen secretes many surface and secretary proteins including various enzymes and pathogenic factors that favour the colonization and infection of host tissue. Alpha-amylase is one of the enzymes secreted by S.aureus which catalyses the breakdown of complex sugars to monosaccharides, which are required for colonization and survival of this pathogen. In the present study we have cloned, sequenced, expressed and characterized α-amylase from the clinical isolate of S.aureus resistant to vancomycin (VRSA). The 25kb plasma DNA was isolated from the clinical isolate of S.aureus and a 600bp PCR amplified product was obtained. The SDS-PAGE analysis of induced and un-induced clone SB3 indicates that α-amylase has a M.W. of 22KD. The rα-amylase was eluted from the gel and enzymes assay was performed. The Km was found to be close to Km of native α-amylase identified in the extracellular and in the cytosolic fraction of S.aureus resistant to vancomycin. The current study clearly indicate that there is clear distinction between the α-amylase of human and different prokaryotes, particularly S.aureus which made this enzyme an ideal target in the development of drugs against infections of tooth and oral cavities.