Sulfapyridine (SPY) is extensively consumed in aquaculture and may cause environmental damages. The mechanism of radicals on the degradation of SPY was still unclear. In this study, Fe²⁺ was found to have a positive contribute on the conversion of sulfate radical (SO₄⁻) to hydroxyl radical (OH) in the peroxydisulfate (PDS) system. Kinetic modeling demonstrated that the concentration of OH was depended on pH, and OH was the dominant radical in Fe²⁺/PDS system. The second-order rates of OH (1.03 × 10¹⁰ M⁻¹ s⁻¹) and SO₄⁻ (8.41 × 10⁹ M⁻¹ s⁻¹) reacting with SPY clarified OH reacted with SPY faster than SO₄⁻. Combining the transformation products (TPs) and quantum chemical calculation, the degradation pathways of SPY by SO₄⁻ and OH were proposed. The antibacterial activity test illuminated that the antibacterial activity of some TPs (TP3 and TP6) was relatively higher than their parent compound. The antibacterial activity of solution was difficult to be removed with the SPY degradation. These results illustrated that the Fe²⁺/PDS treatment need further improvement.