The invention relates to the technical field of fresh keeping of aquatic products and discloses a natural biological fresh-keeping agent for fishes and preparation and application methods thereof. Thenatural biological fresh-keeping agent is prepared from the following components in parts by weight: 14 to 28 parts of a rosemary extracting solution, 6 to 16 parts of a radix clematidis extracting solution, 2 to 10 parts of a spora lygodii extracting solution, 10 to 20 parts of a radix angelicae anomalae extracting solution, 4 to 10 parts of a rhizoma polygonati extracting solution, 2 to 8 partsof a rhizoma curculiginis extracting solution, 6 to 13 parts of a flos chrysanthemi indici extracting solution, 3 to 7 parts of tea polyphenol, 2 to 5 parts of sorbitol, 2 to 5 parts of a pH (Potential of Hydrogen) regulator and 4 to 8 parts of cellulase. Compared with the prior art, the fresh-keeping agent disclosed by the invention can be used for effectively preventing structure damages of muscular tissues of the fishes, serious juice loss and nutrient loss and the like, caused by freezing fresh-keeping, and the food safety problem that the fresh-keeping agent is remained, caused by a chemical fresh-keeping agent, is avoided; the transportation and selling of fresh fishes are ensured; the natural biological fresh-keeping agent is a green, environment-friendly, residue-free, low-cost and safe and reliable fresh-keeping agent with a remarkable fresh-keeping effect.