Methane is one of the major greenhouse gases, recently, the biotechnological conversion from methane to high-value added chemicals have emerged as an effort to reduce methane gas emission. In this study, we optimized ectoine bio-milking conditions in which cells were repeatedly used to improve intracellular and extracellular ectoine yield from methane by using Methylomicrobium alcaliphilum 20ZDP2. First, the cultivation and intracellular ectoine accumulation conditions were optimized with respect to the growth phase and medium salinity to achieve the highest yield of synthesis. Second, ectoine excretion was optimized by determining the ectoine secretion time (15 min) in appropriate medium salinity under hypoosmotic conditions (1% NaCl). Finally, bio-milking of ectoine was successfully repeated more than 10 times using M. alcaliphilum 20ZDP2, and the ectoine yield was improved up to 129.29 mg/ DCW g.