Vesicles composed of amphiphilic block copolymers (i.e. polymersomes) have already been shown to have great potential in drug delivery. Nuclear imaging techniques such as Single Photon Emission Computed Tomography (SPECT) are indispensable in the correct evaluation of biodistribution and pharmacokinetics of newly or not fully investigated polymersome formulations. However, to date, polymer vesicles, in contrast to their lipid counterparts, have not been loaded with radionuclides. In this paper, we have investigated the so-called active loading method to trap radionuclides into preformed polymersomes composed of poly(butadiene-b-ethylene oxide) having variable membrane and brush thickness. We have used tropolone as a lipophilic agent to transport the radioactive isotope of indium, 111In, through the hydrophobic membrane into the aqueous cavity containing the strong hydrophilic chelate diethylene triamine pentaacetic acid (DTPA). The results show that a high loading efficiency of 111In3+ (>85%) can be achieved at short incubation times in polymersomes with membrane thicknesses twice the size of typical lipid bilayers. However, increasing the molecular weight of the block copolymers results in a lower radiolabelling efficiency and a much slower loading rate. In addition, both the DTPA and tropolone concentrations have been found to influence the loading efficiency. Finally, we not only demonstrate that a significant amount of this radioisotope can be successfully encapsulated in the polymersomes, but also report that a negligible loss (<5% in 48 hours) is observed, allowing their safe application in future in vivo studies.