Introduction: In order to search for a new molecule of L-asparaginase with interesting industrial and analytical characteristics, we explored Lake Agulmim, located at 1700 meters’ altitude in Mount Tikjda, part of Mountain range of Djurdjura (Algeria), for the isolation of actinomycete producing strain CA04. Materials and methods: After the molecular identification based in sequencing of 16S rDNA gene of our strain as Streptomyces hydrogenans CA04 and the demonstration of L-asparaginase activity, we extracted the extracellular interest enzyme at 90% ammonium sulphate followed by dialysis and separation by chromatography on Sephacryl S-200 gel. Results: We detected, therefore, two isoforms A and B of MW of 86 and 108KDa, eluted at 32min and 33min respectively, with a total protein level of 0.32mg/ml. An SDS-PAGE control was made showing the existence of the two isoforms with molecular weight mentioned. The L-asparaginase activity was maximal between pH 7 and 8, a temperature of 37°C, for 10min of reaction, with a specific activity of 7.28 IU/mg. On the other hand the activity is stable in the presence of Mg2 +, Cu2 +, Zn+ and EDTA, decreased by Fe3+ and inhibited by Mn+. Finally, the L-asparaginase activity produced by Streptomyces hydrogenans CA04 has a high degree of specificity to the L-Asparagin substrate, with very weak relative activities, against the other nearby substrates, L-Glutamine and L-Aspartic Acid.