Abstract: Hair samples, as a non-invasive biological material, it is gradually gaining attention in the fields of nuclear forensics, environmental monitoring, occupational exposure detection, and medicine due to its certain accumulation effect on human trace elements. At present, in the field of hair analysis, most of the existing researches use the bulk analysis technology to measure the average content of elements in hair, while there are few researches on hair micro-area analysis. In order to expand the application of hair analysis methods, the rat hair samples with high uranium content were obtained by feeding rats with uranyl nitrate solution. The pretreatment method of hair samples and the measurement method of uranium in hair by secondary ion mass spectrometer (SIMS) were studied. The results show that when measuring hair samples using SIMS, the grooves on the surface of the sample target can effectively improve the signal strength stability; coating copper on the sample surface can effectively reduce the surface charge accumulation and reduce the interference of polyatomic ion signal to uranium ion signal. The distribution, average content and isotope ratio of uranium in rat hair were measured by multi collected inductively coupled plasma mass spectrometry (MC-ICP-MS) and SIMS. The results show that this method can effectively judge whether rats drink uranium solution or not, and distinguish the type of uranium taken by rats. This study provides technical support for traceability analysis of hair samples, which is of great significance for the analysis of trace elements in hair.