Alkyl polyglucosides have attracted great research interests as environmental-friendly biological surfactants. Consequently, we investigated the effects of n-hexyl polyglucoside and octyl-decyl polyglucoside on the formation thermodynamics and kinetics of CH4 hydrate, and experimented the separation of CH4/N-2 via hydrates formation with n-hexyl polyglucoside and octyl-decyl polyglucoside, respectively. The results show that both of them could increase the hydrate formation rate and shorten the reaction time. As a result, the separation efficiency of CH4/N-2 via hydrates formation is enhanced significantly. The employ of alkyl polyglucosides in hydrate could promote the development and industrial application of hydrate-based separation method.