L-asparaginase (E.C.220.127.116.11) has been commonly used for the treatment of acute lymphoblastic leukemia in adults and children. It is also used in food industry to reduce acrylamide formation during the preparation of fried food items containing starch at high temperatures. Several microorganisms from the diverse group of bacteria and yeast were reported to be used for L-asparaginase production however, many of the strains also coproduce L-glutaminase which is highly undesirable as it results in cellular stress and neurotoxicity. Thus identification of new sources for the production of glutaminase free L-asparaginase needs to be explored. In this study, we screened endophytic fungi isolated from trees of moist deciduous and semi evergreen forests of the Western Ghats and plants growing in Rono Hills, Arunachal Pradesh, India for the production of glutaminase free L-asparaginase. Using a simple agar plate assay, we found that 33 strains were positive for the L-asparaginase activity among which 19 strains showed glutaminase free L-asparaginase activity. Our results show that: Alternaria sp. endophytic in the leaf of Withania somnifera and growing in the moist deciduous forest of the Western Ghats showed maximum enzyme activity. Optimization of process parameters reveal that maximum L-asparaginase production was observed at 96 h of fermentation and high concentration of glucose in the medium as the carbon source inhibited enzyme production in Alternaria sp. This is the first report on production of glutaminase free L-asparaginase by fungal endophyte Alternaria sp. © 2014 Academic Journals Inc.