A high L-ASNase enzyme production by Bacillus subtilis can hold significant promise as an alternative bacterial source to meet anti-cancer therapies demand. In this study, eight bacterial isolates were obtained from the 35 soil samples of Golestan province, Iran. Samples were collected from different soils and cultured in an M9 medium. Enzyme-producing colonies were distinguished by the color change from yellow to pink. 16s rRNA sequencing identified ASNase-producing bacterial species in soil samples. The presence of L-ASNase in Bacillus subtilis was shown by M9 culture and confirmed by spectrophotometry. Bacillus subtilis was identified by 16S rRNA gene sequencing. L-ASNase isolated from Bacillus subtilis showed effective antioxidant activity and also anti-bacterial activity against Staphylococcus aureus and Escherichia coli. The present study demonstrated the presence of L-ASNase-producing Bacillus subtilis strain in soil samples with antioxidant and antibacterial activity.