Abstract: The accurate determination of ultra-trace plutonium isotopes, particularly ²⁴²Pu, in biological samples is of great significance for occupational internal radiation dose assessment and health monitoring. However, fecal samples present an extremely complex matrix characterized by severe heterogeneity, high organic matter content, and abundant transition metals. These interfering factors severely hinder the accurate quantification of actinides. To address the severe interference from extreme fecal matrices, a highly sensitive quantitative method for the ultra-trace analysis of ²⁴²Pu through the systematic optimization of pretreatment and radiochemical separation conditions was established in this paper. To construct the optimal analytical procedure, a step-by-step systematic optimization was conducted. First, a gradient high-temperature ashing method combined with acid digestion was utilized. The fecal samples were incinerated at optimized gradient temperatures, followed by an aqua regia digestion process to thoroughly destroy the complex organic matrices and insoluble residues. Subsequently, a precise valence adjustment strategy was applied to the digestion solution. A combined redox system consisting of ferrous sulfamate (Fe(NH₂SO₃)₂) and sodium nitrite (NaNO₂) was employed. This specific step was carefully executed to accurately convert and lock the target plutonium nuclide into the tetravalent state (Pu(Ⅳ)). For the radiochemical purification process, extraction chromatography using TEVA resin was utilized. The operational parameters, including the loading acidity and the elution volume, were systematically investigated. Furthermore, a full-procedure methodological validation was designed using multiple independent parallel samples to comprehensively evaluate the batch-to-batch reproducibility. The systematic experimental results confirm the establishment of the optimal analytical flow. Specifically, 600 ℃ is determined as the optimal gradient ashing temperature. For the TEVA resin separation, 3 mol/L HNO₃ provides the optimal loading acidity, and 15 mL is identified as the optimal desorption volume. This specific volume parameter guarantees the highly efficient separation of the target nuclide via TEVA resin while effectively avoiding excessive sample dilution. Furthermore, the full-procedure validation results demonstrate excellent quantitative indicators. The overall chemical recovery of ²⁴²Pu stably distributes in the range of 71.4% to 81.9%. Calculated from the multiple independent tests, the relative standard deviation (RSD) is only 3.8%. In terms of the detection sensitivity, the method detection limit (MDL) is evaluated to be as low as 2.09 fg/g. In conclusion, this systematically optimized method exhibits excellent batch-to-batch reproducibility and strong process stability. It successfully overcomes the severe impacts and interferences caused by the heterogeneous matrix. The proposed analytical strategy provides highly reliable technical support for the precise quantification of actinides in complex biological samples, fully satisfying the stringent requirements for occupational internal exposure monitoring.