The invention discloses a high-freshness and low-bitterness flavor condiment as well as a preparation method and the application thereof. The preparation method comprises the following steps: adding awheat gluten albumen powder and edible yeast into water; then adding glutamine transaminase; insulating and stirring at the temperature of 50 to 55 DEG C for 4 to 5 hours, and enabling the wheat gluten albumen powder and yeast protein to generate a cross-linking reaction; subsequently, adding protease and carrying out insulation enzymolysis for 18 to 24 hours to obtain a yeast and wheat gluten albumen mixed enzymolysis product; concentrating and carrying out spray drying to obtain a flavor condiment powder. According to the preparation method disclosed by the invention, the glutamine transaminase is added to promote wheat gluten albumen protein and the yeast protein to generate the cross-linking reaction and a primary structure of raw protein is changed, so that an original bitter peptidestructure can be eliminated or hidden, and more fresh taste peptide structures can be exposed or generated; by combining with controlled-enzymatic hydrolysis of second-stage protease, bitter peptidecan be hidden to the maximum extent and more fresh taste peptides are released, so that the bitterness of an enzymolysis product (flavor condiment) is effectively reduced and the fresh taste strengthis improved.