Marine fungi have the capacity to extreme environments and   live in a highly competitive ecological position make them suppliers of novel metabolites as L-glutaminase   which is medically useful in the pharmaceutical industry, as an antioxidant and anticancer and antileukemia. This study aimed to isolate and identified some marine fungi and screened for L-glutaminase production and select the most producing marine fungus to optimize the culture conditions for maximizing production of L-glutamenase. The results showed five isolated fungal species; Aspergillus flavus, A. niger, A. terreus, Penicillium digitatum, P. italicum which were identified microscopically according to references books on mycology. The Screening for L-glutaminase production from the isolated fungi utilizing solid and liquid media exhibited that A. flavus out of the isolated fungi, recorded the highest diameter of pink zone (72.3 mm) with using solid medium and highest L-glutaminase activity (11.489U/ml) and highest mycelial dry weight using liquid medium. The optimization culture conditions   of   A. flavus for maximizing L-glutaminase production, showed that the highest L-glutaminase activity and mycelial dry weight were recorded at 30°C for 5 days of incubation, pH6, L-glutamine and fructose as nitrogen and carbon sources. Molecular identification of A. flavus was investigated for confirming the morphomicroscopical identification, Phylogenetic tree based on ITS sequences of rDNA of the isolated fungus closely related to strains accessed from the GenBank. It showed similarity 99.31 -99.83% and 98 - 100% coverage with the strains of A. flavus in GenBank with accession no. NR_111041.