The conventional hydrate promoters and inhibitors, such as tetrahydrofuran (THF), sodium dodecyl sulfate (SDS) and methanol, have the common disadvantages of chemical substances, i.e., pungent flavor, corrosivity, toxicity and anti-degradability. It severely pollutes the environment and limits the application of hydrate-based technologies. Consequently, it is necessary to develop new and green substances as the hydrate promoters or inhibitors. In this work, L-arginine and isooctyl glucoside were used as bio-additives for CH4 hydrate to form bio-hydrate. The effects of L-arginine and isooctyl glucoside on the formation thermodynamics and kinetics of CH4 hydrate were investigated first. Then, the equilibrium separation of CH4/N2 via hydrates formation was conducted in isooctyl glucoside solution. The results show that formation pressure of CH4 hydrate in L-arginine solution is higher than that in pure water at the same temperature. Isooctyl glycosidase can greatly accelerate the hydration rate and increase the gas storage capacity of hydrate. In the separation of CH4/N2, the recovery of CH4 and gas storage capacity of hydrate are significantly increased after adding isooctyl glucoside in water. In conclusion, L-arginine can be used as hydrate inhibitor to replace conventional alcohols in the inhibition of hydrates formation. Isooctyl glycosidase is an effective kinetic promoter of hydrate, which is suitable for CH4 storage in hydrate, and separation of CH4-containing gas mixture via hydrates formation. This work provides two environment friendly bio-additives for hydrate, which will definitely promote the development and application of hydrate research. •We measured formation conditions of CH4 hydrate with two bio-additives, respectively.•The effects of two bio-additives on kinetics of CH4 hydrate were investigated.•The separations of CH4/N2 were conducted in the presence of isooctyl glycosidase.•Green bio-additives of hydrate were developed to replace the chemical additives.