Arsenite oxidase (Aio) plays a central role in the microbial oxidation of As(III) to the generally less toxic and more easily removable As(V). In this study, the aioA and aioB genes from Thiomonas delicata strain DSM 16361 were heterologously expressed in Escherichia coli BL21 (DE3) using the pRSFDuet–1 system, and the recombinant proteins were purified under optimized conditions. The two subunits were purified individually, and their activities were evaluated alone and after reconstitution. AioA displayed markedly higher enzyme activity than AioB, consistent with its role as the catalytic center, whereas purified AioB alone showed no detectable As(III) oxidation activity, supporting its function in intramolecular electron transfer between subunits. When AioA and AioB were combined in a 1:1 molar ratio, the catalytic efficiency (kcat/Km: 1246 ± 26 M⁻¹/s) and Vmax (38 ± 2 µmol/min/mg) increased compared to AioA alone, confirming that AioB enhances electron flux and overall oxidation performance. The purified enzyme exhibited maximum activity at pH 7.0 and 40 °C and retained activity across a broad pH (4–8) and temperature (20–50 °C) range. Activity was largely unaffected by most metal ions and anions tested, although Triton X–100 significantly inhibited catalysis. Batch oxidation tests using purified AioA demonstrated rapid conversion of As(III) under environmentally relevant concentrations: 20 mg/L was fully oxidized within 15 min, and 40 mg/L within 60 min, while higher loadings showed partial conversion within the same reaction time. These findings highlight the potential application of recombinant AioA in enzymatic arsenic remediation and provide a foundation for designing AioA–based biocatalytic treatment systems for contaminated waters.