An acidic peroxidase isoform, POD-A, with a molecular mass of 69.4kDa and an isoelectric point of 3.5 was purified from papaya latex. Using o-phenylenediamine (OPD) as a hydrogen donor (citrate–phosphate as pH buffer), the optimum pH for the function of POD-A was 4.6, and the optimum temperature was 50°C. The peroxidase activity of POD-A toward hydrogen donors was both pH- and concentration-dependent. Under optimal conditions, POD-A catalysed the oxidation of OPD at higher rates than pyrogallol, catechol, quercetin and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). The chemical modification reagents N-bromosuccinimide and sodium azide significantly inhibited POD-A activity. The results of kinetic studies indicated that POD-A followed a ping-pong mechanism and had a Km value of 2.8mM for OPD. Using CPC silica-immobilised POD-A for the determination of micromolar H2O2 in milk, the lower limit of determination was 0.1μM, and the recoveries of added H2O2 were 96–109%.