Abstract: ⁵⁵Fe is an important corresponding activation product during the operation of nuclear power plants. ⁵⁵Fe may be present in the used oil required for main circuit mechanical equipment through cross-contaminating during maintenance activities-including change-out of oil in plant equipment or directing contacting with contaminated water. Currently, used oil from nuclear power plants is mainly stored temporarily at nuclear power sites and needs to be treated by volume reduction to achieve the goal of waste minimization. Evaluation of the content of the important difficult-to-measure beta-radionuclide ⁵⁵Fe is necessary for the management and disposal of used oil. In response to the need for the measurement of ⁵⁵Fe in used oil, a method was established for the analysis of ⁵⁵Fe in used oil. 5 mg of Fe³⁺ was added to the used oil sample, mixed well and left for 1 h to ensure that the Fe³⁺ is in equilibrium with the ⁵⁵Fe in the sample. The iron in the used oil sample was back-extracted to the liquid phase with 9 mol/L HCl, passed through AGMP-1 anion-exchange resin, and the resin column was then rinsed with 60 mL of 12 mol/L HCl followed 8 mL of 6 mol/L HCl to remove the potential interfering nuclides ⁶³Ni, ⁶⁰Co, ⁵⁴Mn, and ⁵¹Cr. Iron was eluted from the anion resin using 3 mL of 0.1 mol/L HCl. For the potentially interfering nuclide ⁵⁹Fe, which cannot be removed by chemical separation, a method for analyzing the liquid scintillation counting spectra of the ⁵⁵Fe and ⁵⁹Fe dual nuclides was established and verified. The verified results show that the method can accurately calculate the ⁵⁹Fe overflowing into the ⁵⁵Fe counting window. By testing three commercially available scintillation cocktails, the acidic sample media containing ⁵⁵Fe mixed with Ultima Gold^TM LLT has the lowest detection limit and highest counting efficiency for LSC measurements of ⁵⁵Fe. After the eluate was measured for iron recovery by an X-ray fluorescence spectrometer, 0.6 mL 14.7 mol/L H₃PO₄ was added to form colourless complex with Fe³⁺. Then, 14 mL of Ultima Gold^TM LLT was added and mixed with the sample. The sample was gently agitated and placed in darkness in the liquid scintillation counter tray, and the counting was started 20 h later to minimize the luminescence. The method was applied to measure ⁵⁵Fe in the used oil, the potential interfering nuclides for ⁵⁵Fe were eliminated by the AGMP-1 anion exchange resin, which the decontamination factors are greater than 10⁵ for ⁶³Ni, ⁵⁴Mn, ⁵¹Cr, and 5651 for ⁶⁰Co. The average recovery of iron is 71%±2%, and the minimum detectable activity concentration for ⁵⁵Fe in used oil is 0.02 Bq/g for 10 g sample with accounting time of 1 h. The method was validated by using spiked samples, and the measured ⁵⁵Fe results agree well with the expected ⁵⁵Fe values within an acceptable deviation of −2.9%-6.0%.