Objective: Improving the accuracy of the determination of fat in CMC-Na fermented milk. Methods: The national standard method-alkali hydrolysis method was used to determine the fat contained in CMC-Na fermented milk. By optimizing the key parameters in the alkali hydrolysis method, hydrolysis of hydrolysis instead of ammonia hydrolysis, and adding metal ions to the alkali hydrolysis method, the suitable fat containing CMC-Na fermented milk was explored. Results: ① With the increase of CMC-Na content, the lower the fat result in CMC-Na fermented milk measured by alkaline hydrolysis; ② The change of the four key conditions, such as the ammonia volume, hydrolysis time, hydrolysis temperature and extraction frequency in alkaline hydrolysis, will not affect the determination on fat in CMC-Na fermented milk. ③ When 2 mL hydrochloric acid hydrolysis was used to instead ammonia hydrolysis, the fat determination results in fermented milk with different CMC-Na content (0.50%, 0.75%, 1.00%) were almost the same as the alkali hydrolysis method for the determination of CMC-Na-free fermented milk; ④ When metal ions Na⁺, K⁺ and Ca²⁺ were added to the alkali hydrolysis method, the higher the concentration of Na⁺ and K⁺, the higher the fat determination results containing CMC-Na fermented milk, and the normal value will be achieved after the concentration of metal ions reaches to 0.5 mol/L, while the increase of Ca²⁺ concentration, and the fat determination will be determined. As a result, it increased slightly first, and then decreased significantly. Conclusion: The addition of CMC-Na will make the fat determination results in the fermented milk low. Hydrochloric acid hydrolysis or Na⁺ and K⁺ can be added to the alkali hydrolysis method to improve the accuracy of fat determination in the fermented milk containing CMC-Na.