The invention discloses a bio-based polyol as well as a preparation method and application thereof. The preparation method comprises the following steps: (1) simultaneously pumping a mixed solution prepared by dissolving pentaerythritol and styrene oxide in an organic solvent and an alkali solution into a first microreactor of a microchannel modular reaction device for reacting to obtain a reaction effluent containing polyol; and (2) simultaneously pumping a mixed solution prepared by dissolving epoxy vegetable oil in an organic solvent and the reaction effluent containing polyol prepared in the step (1) into a second microreactor of the microchannel modular reaction device for reacting to obtain the bio-based polyol. The preparation method provided by the invention is a continuous flow process, easy to operate, few in process step, short in reaction time, low in energy consumption, cheap and easily available in raw material, recyclable in organic solvent, relatively green and environmentally friendly, few in side effect and high in product quality.