β-1,3-1,4-glucanase (β-glucanase) is an enzybiotic enzyme that cleaves linear β-glucans with β-1,3 and β-1,4 linkage, including cereal β-glucans. It has several applications in industry, agriculture, and medicine in which it has been observed with a broad antimicrobial spectrum. A local marine strain with high activity of β-glucanase was screened and isolated from Gabal El-Zeit offshore in the Red Sea of Egypt. It was successfully identified through a partially 16S rRNA gene sequencing technique as Halomonas meridiana ES021. The medium was optimized using a multifactorial matrix of Plackett-Burman design, and the most influencing components were barley flour, urea and KH₂PO₄. The enzyme activity was positively affected by barley flour, urea and negatively affected by KH₂PO₄. The optimal medium obtained through statistical design increased enzyme activity by 1.4-fold from 455.83±20.45 to 597.50±24.50U/ mL. In addition, a Box-Behnken experimental design was studied to investigate interactions between barley flour, urea and KH₂PO₄ concentrations. In the optimized medium, the enzyme activity was increased by about 1.8-fold from 597.50±24.50 U/mL to 1095.50±31.00 U/mL. Moreover, cell immobilization by adsorption and entrapment was investigated and immobilized cultures of Halomonas meridiana ES021, which were entrapped in a 3% agarose gel produced the maximal β-glucanase activity, which was about 1111.67 ± 29U/ mL,  followed by cultures adsorbed on clay particles, which was about 916.97 ±29.47U/ mL.