L-asparaginase is an enzyme included in the treatment of acute lymphoblastic leukemia (ALL). Its thrabiutic mechanism is the hydrolyzes of l-asparagine, which is essential amino acid for neoplastic cells while not essential and can be synthesized by normal cells. The proplem of l-asparaginase from microbial origen represented in its association with glutaminase activity. The search for new eucarutic l-asparaginase-producers among microorganisms with low glutaminase affinity is important. This study investigated the biotechnological potential of filamentous fungi isolated from the cogongrass (Imperata cylindrical) rhizosphere soil. Fifteen fungal species were isolated from 12 environmental samples, and enzyme production was measured using a plate assay, followed by cell disruption to determine l-asparaginase release after submerged fermentation. L-asparaginase free of glutaminase-producing fungus was identified as Penicillium sp. by molecular-based methods. According to optimization results for enzyme production, asparagine at 15 g/L-1 and sucrose at 5 g/L-1 were the best sources of C and N, respectively, along with pH 7, 35 °C, 75 g/L-1 of NaCL, and 0.1 g/L-1 of Fe2+ under shaking conditions at 150 rpm for 4 days.