A method for low-level, low-potential electrochemical detection of hydrogen peroxide using a chemically activated redox mediator is presented. This method is unique in that it utilizes a mediator, Amplex Red, which is only redox-active when chemically oxidized by H2O2 in the presence of the enzyme horseradish peroxidase (HRP). When employed in concert with microelectrode square wave voltammetry to optimize sensing at ultralow concentrations (< 1 μM), this method exhibits marked improvements in analytical sensitivity and detection limits (limit of detection as low as 8 pM) over existing protocols. Sensing schemes incorporating both freely diffusing and immobilized HRP are evaluated, and the resulting analytical sensitivities are 1.22 ± 0.04 and (2.1 ± 0.6) × 10-1 μA/(μM mm2), respectively, for peroxide concentrations in the high picomolar to low micromolar range. A second linear region exists for lower peroxide concentrations. Furthermore, quantitative enzyme kinetics analysis using Michaelis-Menten parameters is possible through interpretation of data collected in this scheme. Km values for soluble and immobilized HRP were 84 ± 13 and 504 ± 19 μM, respectively. This method is amenable to any biological detection scheme that generates hydrogen peroxide as a reactive product.