A puncture-proof rain boot comprises a sole, a vamp and a shoe tube, the vamp and the shoe tube are integrally molded, the vamp and the shoe tube respectively have a three-layer structure and respectively concretely comprise a waterproof layer, a buffering and puncture proofing layer and a lining foot protection layer from outside to inside, above three layers are sequentially bonded together, the waterproof layer is made of rubber or a PVC material, and the buffering and puncture proofing layer is made of a puncture-proof high toughness material and prevents pointed hard objects from puncturing the vamp; the buffering and puncture proofing layer is made from, by weight, 30-60 parts of nylon6, 20-40 parts of short glass fibers, 1-1.2 parts of dimethicone, 0.5-1 part of an antioxidant, 5-10 parts of linear polyethylene, 3-5 parts of polylactic acid, 1-5 parts of a synergistic agent, 0.5-1 part of a nucleating agent, 0.5-1 part of an ultraviolet ray absorber, 0.5-1 part of a surfactant, 1-3 parts of a chain extender, 0.5-1 part of dry oxhide gelatin powder and 0.5-1 part of ceramsite nanopowder; and the lining foot protection layer is formed by blending polyester fibers, bamboo fibers and man-made spider fibers, and the lining foot protection layer has good elasticity and toughness, allows skins in contact with the lining foot protection layer to be comfortable, and has an antibacterial effect.