To detect a target protein in biological samples, a fusion protein was designed composed of a peroxidase from <i>Arthromyces ramosus</i> (ARP) and parts of the antibody-binding domains of <i>Staphylococcus aureus</i> protein A and <i>Streptococcus</i> protein G (PG). The ARP-PG fusion protein was successfully expressed by a heterologous protein expression system in <i>Brevibacillus choshinensis</i>. The fusion protein was secreted as an active form in culture media. The production of ARP-PG with higher peroxidase activity was observed by the addition of 5-aminolevulinic acid to the culture media. The performance of purified ARP-PG was validated by dot blotting for the detection of transferrin as a model target protein. A comparable performance in the dot blot analysis was attained using a culture supernatant containing crude but active ARP-PG, indicating the practicality of the <i>Brevibacillus</i> protein secretion system.