Abstract-Microbial α-amylase is a highly demanded industrial enzyme with extensive commercial applications in various sectors. Studies were carried out with a bacterial strain producing extracellular α-amylase, isolated from rhizospheric soil of Euphorbia hirta. The isolate was gram positive, motile rod, bearing terminal endospore. It exhibited >98% similarity with the reference strains in the GenBank. The phylogenetic tree constructed on the basis of 16S rRNA gene sequences revealed that it clustered with the closest members of Bacillus subtilis and identified as Bacillus subtilis KC3. Furthermore, the effects of incubation period, temperature, pH, different carbon and nitrogen sources, metal salts and different substrate concentrations of the medium were optimized. The maximum enzyme production was found after 48 h (22.92 U/ml) of incubation at temperature 40°C and pH 7. The optimal temperature and pH for enzyme activity were 50°C and 6.5 respectively. Barley starch (27.27 U/ml) was observed to be the best inducer followed by corn starch (24.30 U/ml) and maltose (19.10 U/ml). The addition of glucose to the culture medium greatly reduced the synthesis of α-amylase (5.45 U/ml) which demonstrates that a classical glucose effect is operative in this organism. The effects of different metal ions (Ca 2+, Fe 2+ , Mg 2+ , Zn 2+ and Cu 2+ ) on amylolytic activity were investigated and it was found that 0.1% of Ca 2+ increased enzyme production (28.83U/ml), whereas other metal ions exhibited inhibitory effects. The enzyme production was maximum at 2% substrate (starch) concentration, which declined beyond it. These characteristics of Bacillus subtilis KC3, suggest that this is a promising isolate which merits further investigations for potential applications in various biotechnological processes.Index Terms-α-amylase, bacillus subtilis KC3, euphorbia hirta, rhizospheric soil