L- asparaginase is an enzyme which is used in food industry and also as chemotherapeutic agent against the acute lymphoblastic leukemia and lymphosarcoma. L-asparaginase is a wide spread enzyme found in many plants, Animals, bacteria, fungi and algae. The present study was aimed at isolating fungal strains capable of producing L- asparaginase from soil samples of karaikudi, Sivaganga district. The fungal isolates were isolated from soil samples and screened for L- Asparaginase production using modified czapekdox agar medium which contain L- Asparagine as main source of carbon and phenol red indicator. On the basis of pink colour zone formed, positive fungal strains were selected. Quantitative enzyme assay was performed by measuring the ammonia liberated by Nesslerization. The highest producer was identified as Aspergillus species.