Oxytetracycline (OTC) fermentation residue (OFR) is biosolid waste of OTC production process. To date, the main treatment of OFR is incineration, and the study regarding its other harmless disposal and resource utilization attracts increasing attention. In this study, soil incubation was performed to investigate the influence of Fenton oxidation treated OFR (FOFR) on soil physicochemical properties, enzymatic activity, and microbial community. Further, antibiotic residue, antibiotic resistance genes (ARGs), and mobile genetic elements (MGEs) were determined and analyzed in the OFR-amended soil. After adding FOFR, soil pH increased firstly before 20 days and then decreased. The contents of soil organic matters, nitrogen, phosphorus, and potassium of FOFR addition treatment were higher than those of OFR one indicating that Fenton oxidation could promote the availability of these nutrients in OFR. The enzyme activity was not inhibited throughout the incubation stage of FOFR-amended soil. In the soils, OTC concentration decreased quickly, below the detection limit of LC/MS/MS after 60 days, and Fenton oxidation could significantly eliminate residual OTC of OFR. Bacterial community structure of FOFR-amended soil was converged to be similar with the control soil after 90 days. Our results suggested that Fenton oxidation might be in favor of limiting the spread of OTC resistance genes and MGEs of OFR in soil reclamation.