The peroxidase-like activity of dissolved Fe₃O₄ magnetic nanoparticles (MNPs) was determined and compared with whole Fe₃O₄ MNPs in this study. We detected the peroxidase-like activity of Fe₃O₄ MNPs through the catalytic reaction between Fe₃O₄ MNPs and TMB. Due to the high catalytic activity of ferric ions and ferrous ions released from Fe₃O₄ MNPs after dissolution, dissolved Fe₃O₄ MNPs showed much better peroxidase-like activity than whole Fe₃O₄ MNPs. In addition, we used the peroxidase-like activity of Fe₃O₄ MNPs in the diagnosis of mycoplasma pneumonia infection in which Fe₃O₄ MNPs was used as substitution of horseradish peroxidase. Both Fe₃O₄ MNPs modified with amino groups (MNPs-NH₂) and Fe₃O₄ MNPs modified with polyethyleneimine (MNPs-PEI) were successfully used in the indirect immunoassay for the detection of mycoplasma pneumoniae. And the results of dissolved Fe₃O₄ MNPs were more sensitive and specific than that of whole Fe₃O₄ MNPs.