The invention relates to preparation and a using method of a biomass-based additive for improving diesel performance. A preparation method of the additive comprises the following steps of utilizing a pyrolyzing furnace to quickly warm under inert atmosphere to pyrolyze biomass, dewatering and filtering a pyrolysis product of water-phase bio oil to obtain bio oil, performing esterification reaction on the bio oil, evaporating and condensing short-chain ester generated by the esterification reaction to obtain short-chain ester solution, namely the biomass-based additive for improving diesel performance. The using method of the additive comprises the following steps of adding the biomass-based additive into diesel fuel and evenly stirring to obtain performance-improved short-chain ester-diesel mixed fuel. In the short-chain ester-diesel mixed fuel, the volume ratio V% of the short-chain ester is larger than 0% and smaller than or equal to 50%. The method can efficiently collect the short-chain ester solution, improves mixing proportion of the additive in the diesel mixed fuel and reduces petroleum resource use.