The analysis of Pu isotopes with radiometric or mass spectrometry techniques requires prior chemical separation to overcome interferences from other actinides and to remove matrix components. These separations are usually carried out using extraction or ion exchange chromatography. In this work, flow electrolytic separation on anodized carbon fibers is explored as a new alternative approach for the separation and analysis of Pu isotopes by means of radiometric methods. A high-surface-area carbon fiber felt electrode was anodized and used for flow electrolytic accumulation and release of Pu. Characterization of the anodized carbon fiber felt was carried out using X-ray photoelectron and infrared spectroscopy. The conditions needed for the retention of Pu during electrolysis were investigated, optimized, and used to develop a method for the measurement of ²³⁸Pu or ^239,240Pu by α-spectrometry. This method was evaluated with digested solid samples (i.e., wipe test, ceramics, and sludge) and compared with traditional chromatographic separation approaches. It was found that oxygen-containing functional groups are introduced on the carbon fiber surface upon its anodization. This allows the accumulation of Pu(IV), which is produced by adjusting the electrode potential and can be released by electroreduction to Pu(III), whereas other actinides (e.g., U, Am, and Cm) as well as matrix components are not retained. This provides a fast, single-step separation of Pu, free of impurities from reagents or resins, which may be detrimental to the preparation of α-sources. The successful measurement of Pu isotopes confirmed the reliability and good tolerance of this approach for highly complex matrices.