The invention discloses quick-frozen and fresh-keeping thousand-layer Tofu and a preparation method of the quick-frozen and fresh-keeping thousand-layer Tofu. The quick-frozen and fresh-keeping thousand-layer Tofu comprises the following components in parts by weight: 13-15 parts of soy isolate protein, 0.1-0.25 part of glutamine transaminase, 2-3 parts of cassava acetate modified starch, 5-8 parts of soybean oil, 0.3-0.6 part of aginomoto, 0.3-0.6 part of white sugar, 0.4-0.7 part of complex adhesive, 2.5-3.5 parts of duck egg white and 68-75 parts by weight of water. The quick-frozen and fresh-keeping thousand-layer Tofu disclosed by adopting the preparation method is prepared from the raw materials of the soy isolate protein, the glutamine transaminase, the cassava acetate modified starch, the soybean oil, the aginomoto, the white sugar, the complex adhesive and the duck egg white, keeps the original tender and fresh taste of Tofu, has a special tender, crisp, tasty, delicious taste, and has better elasticity and toughness, and strong soup water absorbing capacity; and regardless of frying, stir-frying, oil-frying, stewing or blending other seasonings, the quick-frozen and fresh-keeping thousand-layer Tofu is special in flavor and delicious in taste when being eaten.