This article reports the synthesis and use of surface-modified iron oxide particles for the simultaneous purification and immobilization of Bacillus stearothermophilus aminopeptidase II ( BsAPII) tagged C-terminally with either tri- or nona-lysines ( BsAPII-Lys3/9). The carboxylated magnetic particles were prepared by the simple co-precipitation of Fe3+/Fe2+ in aqueous medium and then subsequently modified with adipic acid. Transmission electron microscopy (TEM) micrographs showed that the carboxylated magnetic particles remained discrete and had no significant change in size after binding BsAPIIs. Wild-type enzyme and BsAPII-Lys3 could be purified to near homogeneity by the carboxylated magnetic particles, but it was not easy to elute the adsorbed BsAPII-Lys9 from the matrix. Free BsAPII, BsAPII-Lys3, and BsAPII-Lys9 were active in the temperature range 50–70°C and all had an optimum of 50°C, whereas the optimum temperature and thermal stability of BsAPII-Lys3 and BsAPII-Lys9 were improved as a result of immobilization. The immobilized BsAPII-Lys9 could be recycled ten times without a significant loss of the enzyme activity and had a better stability during storage than BsAPII. Owing to its high efficiency and cost-effectiveness, this magnetic adsorbent may be used as a novel purification-immobilization system for the positively charged enzymes.