Pyrogallol red (PGR) was identified as a novel optical probe for the detection of hydrogen peroxide (H2O2) based on horseradish peroxidase (HRP)-catalyzed oxidation. Response surface methodology (RSM) was applied as a tool to optimize the concentrations of PGR (100 µmol L−1), HRP (1 U mL−1) and H2O2 (250 µmol L−1) and used to develop a sensitive PGR-based catalase (CAT) activity assay (PGR-CAT assay). N-ethylmaleimide -NEM- (102 mmol L−1) was used to avoid interference produced by thiol groups while protecting CAT activity. Incubation time (30 min) for samples or CAT used as standard and H2O2 as well as signal stability (stable between 5 and 60 min) were also evaluated. PGR-CAT assay was linear within the range of 0–4 U mL−1 (R2=0.993) and very sensitive with limits of detection (LOD) of 0.005 U mL−1 and quantitation (LOQ) of 0.01 U mL−1. PGR-CAT assay showed an adequate intra-day RSD=0.6–9.5% and inter-day RSD=2.4–8.9%. Bland-Altman analysis and Passing-Bablok and Pearson correlation analysis showed good agreement between CAT activity as measured by the PRG-CAT assay and the Amplex Red assay. The PGR-CAT assay is more sensitive than all the other colorimetric assays reported, particularly the Amplex Red assay, and the cost of PGR is a small fraction (about 1/1000) of that of an Amplex Red probe, so it can be expected to find wide use among scientists studying CAT activity in biological samples.