Fluorine-containing fluoroquinolones are cumulative toxins and pose a grave threat to health of animals and human beings, and an effective and feasible strategy to the defluorination by breaking C-F bond to decrease toxicity is in an urgent requirement. Herein, Ti(III)-laden TiO2 nanotube arrays (RTNA) were deliberately fabricated for electrochemical degradation and defluorination of levofloxacin (LVF). Characterizations verified that Ti(III) and oxygen vacancies were generated on RTNA via electrochemical reduction of TiO2 nanotube arrays (TNA). The degradation kinetics of LVF on RTNA (0.85 h−1) was increased by about 15 times as compared with that on TNA (0.06 h−1); the corresponding degradation and defluorination efficiencies by RTNA were also dramatically increased. Moreover, the effect of electrolyte on the defluorination efficiency suggests the significant role of sulfate; that is, abundant sulfate radical (SO4-•) was generated in the media of sodium sulfate during the electrochemical oxidation process, and it served as the dominant radical to break C-F bond for the defluorination of LVF, whereas it is not the case for hydroxyl radical (•OH) in sodium carbonate. The pathways and mechanisms of degradation and defluorination in electrolyte of sodium sulfate or carbonate were proposed and further confirmed the difference in the electrolyte-dependent degradation mechanisms.