The present disclosure relates to a pyrolysis bio-oil fractional condensation device and method capable of cooling medium self-circulation. The device includes a primary condensation system, a secondary condensation system and a cooling medium self-regulation heat exchange system. The primary condensation system uses the temperature-regulated cooling medium to condense the macromolecular tar by direct heat exchange with the pyrolysis volatiles. The condensed tar is heated, pushed and scraped with a rotary mechanism to prevent adhesion. The spray liquid in the secondary condensation system exchange heat with the uncondensed volatiles directly for secondary condensation. The cooling medium self-regulation heat exchange system realizes self-circulation and self-balance of the cooling medium mass flow and energy flow by integrating heat absorption during biomass raw material feeding and drying, heat release during volatiles condensation, and heat absorption during pyrolysis char cooling, and realized the independent operation of the condensation device in the mobile biomass pyrolysis system.