Objective To establish a method for determination the fat content in flavored fermented milk containing thickeners by papain-gravimetric method. Methods Papain was used to reduce the adsorption of fat by casein in dairy products. The amount of papain added was 0.5 g, the enzymatic hydrolysis temperature was 65 ℃, and the hydrolysis time was 20 min. A mixture of ether and petroleum ether with equal volumes [25:25 (V:V)] was used to extract fat twice from the flavored fermented milk hydrolysate containing thickening agents. The extraction solvent was evaporated in a water bath, and the fat content was quantified by gravimetric method after drying in an oven. Results The determination of fat content in flavored fermented milk with thickener flavor was consistent with the label value, with a relative standard deviation of 0.40% and a maximum deviation of 0.05 g/100 g, which met the precision requirements of GB 5009.6—2016 third method for determination of fat in foods. Conclusion This method is simple to operate and conform to the concept of green analytical chemistry, compared with GB 5009.6— 2016 third method, it reduces the total volume of mixed ethers used, thereby minimizing harm to human health and environmental pollution. With good reproducibility, it can effectively determine the fat content in flavored fermented milk containing thickeners, providing robust technical support for quality control of such products.