Large amounts of uranium-containing radioactive waste are generated in reactor operation and in the research and manufacture of nuclear fuel elements. At present, there are various uranium enrichment and separation methods such as reduction precipitation method, ion exchange method, solvent extraction method, membrane filtration method, adsorption method, and microorganism method to treat the uranium-containing radioactive waste generated in the related processes of nuclear facilities. However, the airborne effluent or liquid effluent discharged after treatment may still contain radioactive uranium. It is well known that uranium is a radioactive heavy metal element, and its radioactive and chemical toxicity cannot be ignored. Uranium in the environment enters the human body through the food route, and its long half-life can make the human body suffer from continuous radioactive internal radiation damage. As an environmental medium, organisms are closely related to the entry of uranium into the human body through food. Therefore, it is of great significance to carry out accurate measurement of uranium content in environmental-grade biological samples around nuclear facilities, however, complete and accurate measurement results include measurement data and uncertainty. Laser fluorescence method is a method for rapid analysis of uranium content in environmental samples. It has the advantages of high sensitivity, simple sample pretreatment, and wide measurement range, which has been widely used in nuclear industry, environmental monitoring and scientific research. At present, there is a lack of relevant reports on the uncertainty of the measurement of total uranium content in environmental-grade biological samples by laser fluorescence method. It is of great significance to accurately measure the uranium content in biological samples by evaluating the uncertainty of this method. In this paper, the WGJ-III trace uranium analyzer was used to analyze the uncertainty source of total uranium in environmental-grade biological samples by laser fluorescence method. The uncertainty measurement model was established, the uncertainty components were quantified, and the expanded uncertainty of the measurement of total uranium content in environmental biological samples was calculated. The evaluation results showed that the expanded uncertainty of a 0.05 g environmental biological sample is 10.8% (k = 2) without dilution, and the dominant uncertainty component is derived from the measurement uncertainty of sample fluorescence counting.