Abstract Out of 210 actinomycetes isolates belonged to the genus Streptomyces recovered from rhizospheric soil of Triticum vulgaris, Zea mays and Vicia faba cultivated in Assuit and New Valley Governorates of Egypt, ten isolates were capable of producing high amounts of L-glutaminase enzyme. The most potent L-glutaminase producer isolate was Streptomyces sp. ASU319 recovered from rhizosphere of Triticum vulgaris. The higher producer actinomycete isolate was identified by sequencing of 16S rRNA as Streptomyces variabilis ASU319 and was deposited in the GenBank nucleotide sequence database under accession number KC145278. Factors affecting L-glutaminase production by the Streptomyces variabilis ASU319(KC145278) were examined and the results revealed that the maximum L-glutaminase value was obtained when the isolate cultivated in the production broth medium supplemented by glutamine 8 mg/ml, adjusted at pH 4 and incubated at 35 °C. These results suggest that L-glutaminase-producing Streptomyces variabilis ASU319 could be used as a plant growth promoting rhizobacteria by increasing the ammonia content in the rhizosphere soil across degradation of the agricultural wastes. Also this isolate can be used in both pharmaceutical and food industrial application for L-glutaminase production on commercial scale.