Composition, morphology, and surface characteristics of solid substrates play critical roles in regulating immobilized enzyme activity. Grapheme oxide (GO), a novel nanostructured material, has been illustrated as an ideal enzyme immobilization substrate due to its unique chemical and structural properties. Physical properties and catalytic activity of GO immobilized horseradish peroxidase (HRP) and its application in phenolic compound removal are described in the present study. HRP loading on GO was found to be much higher than that on reported substrates. The GO immobilized HRP showed improved thermal stability and a wide active pH range, attractive for practical applications. The removal of phenolic compounds from aqueous solution using the GO immobilized HRP was explored with seven phenolic compounds as model substrates. The GO immobilized HRP exhibited overall a high removal efficiency to several phenolic compounds in comparison to soluble HRP, especially for 2,4-dimetheoxyphenol and 2-chlorphenol, the latter a major component of industrial wastewater.