The invention discloses a method for preparing a sea squirt nanocrystalline cellulose/rubber nanocomposite with a one-pot process. According to the method, natural latex is diluted at the room temperature, and an emulsifier is slowly dropwise added; a sea squirt nanocrystalline cellulose suspension and emulsified natural latex are blended, and a premix is obtained; formic acid is slowly dropwise added to the premix, and hydrogen peroxide is slowly added for a reaction; after the reaction ends, the pH value is adjusted to 6-8, solidification, blanching, dewatering and drying are performed, and a sea squirt nanocrystalline cellulose/rubber mixture is obtained; the mixture and a vulcanizing agent are mixed in an open mill and left for vulcanizing formation. The problem of poorer strength due to physical interaction of two interfaces in the prior art is solved, and the prepared rubber nanocomposite has excellent properties such as high strength, high modulus, high wear resistance and the like and can more sufficiently play the strengthening effect of nanocrystal cellulose.