The pharmaceutical factories of oxytetracycline (OTC) massively produce OTC fermentation residues (OFRs). The high content of residual OTC and antibiotic resistance genes in OFRs must to be considered and controlled at an acceptable level. This study therefore investigated the applicability of Fenton oxidation in OTC degradation and resistant gene inactivation of OFRs. The results revealed that Fe2+ as catalyzer could very rapidly activate H2O2 to produce HO•, leading to instantaneous degradation of OTC. The optimum conditions for OTC removal were 60 mM H2O2 and 140 mg/L Fe2+ under pH 7. After Fenton oxidation treatment, the release of water-soluble polysaccharides, NO3–N, and PO4–P was enhanced, whereas for proteins and NH3–N were reduced. Three soluble fluorescence components (humic, tryptophan-like, and humic acid-like substances) were identified through fluorescence spectra with parallel factor analysis, and their reduction exceeded 50% after Fenton oxidation. There were twelve intermediates and three degradation pathways of OTC in OFRs during Fenton process. According to toxicity prediction, the comprehensive toxicity of OTC in OFRs was alleviated via Fenton oxidation treatment. In addition, Fenton oxidation showed the ability to reduce antibiotic resistance genes and mobile genetic elements, and even tetO, tetG, intI1, and intI2 were eliminated completely. These results suggested that Fenton oxidation treatment could be an efficient strategy for removing OTC and resistance genes in OFRs.