Mercury speciation is of significant importance in environmental and biological analysis because its toxicity and metabolic behavior in the human body differ among species. Nanomaterial-assisted optical sensors are widely used for mercury ion detection but rarely applied in mercury speciation analysis. In this work, we develop a novel colorimetric sensing strategy for mercury speciation based on mercury-stimulated peroxidase mimetic activity of gold nanoparticles with the assistance of different reductants. In the presence of a weak reductant, only inorganic mercury can be reduced to Hg⁰, whereas both inorganic mercury and organic mercury can be reduced to Hg⁰ in the presence of a strong reductant. Due to the high affinity between Hg and Au, Hg⁰ deposits on the AuNP surface in the form of a Au–Hg amalgam, leading to a remarkable enhancement of peroxidase mimetic activity of gold nanoparticles. On the basis of this effect, inorganic mercury and total mercury can be detected by using 3,3′,5,5′-tetramethylbenzidine (TMB) as the substrate. The limits of detection for inorganic mercury and total mercury are 1.9 and 0.9 nM within 5–100 nM, respectively. The selectivity of this sensing system is high due to the specificity of Au–Hg interaction. Its practical applications are further demonstrated by organic mercury analysis in a fish sample and mercury speciation in a human hair sample.