A total of fifty six actinomycete isolates were isolated from the ten marine sediments of south India. Marine environment is a potential source of novel actinomycetes, which are a potent source of antibiotics and novel bioactive compounds. The isolates were identified as actinomycetes by morphological, biochemical and microscopic studies. The isolated actinomycetes were screened for L-asparaginase activity by rapid plate assay method. Based on screening, isolate 1, 2 and 18 were showed large clear pink zone and its diameter (dm) was measured as 9.0, 8.5 and 8.5 cm and the enzyme production has been determined by nesslerization method. The spectrophotometric assay of enzyme activity of the isolates 1, 2 and 18 were found to be 1.92, 1.48 and 1.46 U/ml.