A new simple, rapid, and efficient methodology to produce undamaged graphene sheets from graphite flakes in water by a bio-exfoliation technology is described. The methodology consists in the application of a lipase, with a very exclusive mechanism of interaction with hydrophobic surfaces, combined with a previous mechanical sonication, to selectively generate biographene in water. The adsorption of the lipase on the graphene sheets permits to keep the sheets separated in comparison with other methods. It is possible to obtain more than 80% of graphene (in the form of Few Layer Graphene) from low-cost graphite and with less damage compared to commercial graphene oxide (GO) or reduced GO. Experimental analysis demonstrated the formation of bilayer graphene mainly using lipase from Thermomyces lanuginosus (TLL).