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More and more recent literatures have shown that iron and copper, especially iron plays critical roles in the initiation and propagation of wine oxidation, which seems to depend not only on the total concentration of iron, but also more on the speciation of iron in wine. A rapid method for directly accurate determination of total iron and ferrous ion (Fe2+) in wine was developed by optimizing the ferrozine-based assay for total iron detection, and the effects of ferric ion (Fe3+) and ferrozine levels on the quantification of Fe2+ under wine conditions were also investigated. The results showed that the modified ferrozine assay was established for the direct determination of total iron in wine, the standard curve exhibited a good linear relationship between absorbance and iron concentration from 0.25mg/L to 2.00mg/L (R2=0.9979) and the method detection limit was 0.0115mg/L, with the recovery rate of 94.31%~104.34%. Moreover, the total iron concentration determined by the modified ferrozine assay was in good accordance with that measured by the atomic absorption spectroscopy (AAS) in different wine samples. However, the presence of Fe3+ seriously interfered with the accurate measurement of Fe2+ in wine, and the optimized molar ratio of ferrozine to total iron was 7, with fitting accuracy ranging from 101.98% to 113.50% for Fe2+ determination. The assay was applied to some commercial bottled wines, and it was confirmed that Fe2+ was the dominant iron in wine, although the percentage of Fe2+ in red wines was much higher than that in white ones. In brief, the modified ferrozine assay was simple, accurate and reliable to determine total iron and Fe2+ in wine rapidly, and it can be widely used in the wine industry in future.