The influence of a biodegradable agent, N,N-bis(carboxymethyl)glutamic acid (GLDA), on chloramphenicol (CAP) degradation in pyrite/H2O2 system was investigated. The results indicated that the presence of GLDA could extend the working pH range and provided favorable performance at alkaline conditions. The addition of 100 μmol/L GLDA increased the CAP removal rate to 83.3% at pH 8 due to the reinforced formation of radical dotOH. The beneficial effect was found to be indifferent of possible reduction of redox potential (Fe(III)/Fe(II)), probable decrease of reaction pH and complex of structured iron on pyrite surface. However, it was correlated well with the facilitated autocatalytic oxidation reaction by Fe(III)-GLDA that promoted cycle of iron, which was confirmed by the determination of ESR and sulfate ion leaching. Based on GC–MS results, the presence of GLDA led to further oxidation of product with reinforced oxidation efficiency, and the potential degradation routine remains similar. All these provide some fundamental insights into the characteristics of environment friendly GLDA-enhanced pyrite/H2O2 system for bio-refractory contaminants removal under neutral or alkaline conditions, and enrich the theoretical knowledge of heterogeneous Fenton-like processes.