The invention discloses a preparation method and a thixotropy test method of nano calcium carbonate with negative thixotropy, and the preparation method includes (1) calcining limestone at 800-1000DEG C to obtain calcium oxide, adding water to digest, sieving to remove coarse particles, refining into calcium hydroxide emulsion; (2) adjusting the calcium hydroxide emulsion concentration and temperature, then sending into a bubbling carbonation tower, leading in certain concentrations of carbon dioxide for bubbling carbonation, carbonating to the pH value to be less than 7 for carbide carbonation end point, selecting calcium carbonate boiled pulp with the dry powder specific surface area of 25 m<2> / g-40 m<2> / g; (3) sending the calcium carbonate boiled pulp into an activation reaction tank, adding a certain proportion of medium and long chain fat fatty acid saponification liquid for wet activation treatment; and (4) filtering by pressing, drying and crushing the activated activation solution, sending into a high-speed mixer, adding a certain proportion of hydroxy fatty acids for dry activation for a certain time at the temperature of 50-70DEG C, and after the activation packing to obtain the nano calcium carbonate with the negative thixotropy.