The growth of dendrite is the major limitation to the development of the Li-metal battery. To solve it, we disclose the preparation and performances of separator (MAGly) with a complete “green” formulation using biosourced and sustainable compounds: agarose as biopolymer along with glycerol as plasticizing agent. The natural biopolymer films are non-porous in nature and possess high elasticity with high stiffness along a wide temperature range (−35 to 180 °C), able to prevent the perpendicular dendritic Li growth. Moreover, they provide high Li+ ionic conductivity, which was evident from electrochemical symmetrical battery tests resulted in efficient plating/stripping of Li metal, without dendrite formation. Preliminary tests in Li battery, with LiFePO4 as positive electrode show very satisfying performance regarding the same test with the commercial Celgard® separator. Furthermore, the application of this new sustainable separator can be extended to post Li-metal system as demonstrated by the electrochemical tests realized with K+/K.