The development and optimization of separation and purification methods for high-purity iopamidol were performed based on preparative high-performance liquid chromatography (prep-HPLC). In this study, a reversed-phase separation method for the analysis of iopamidol was developed first. The effects of chromatographic parameters, including two kinds of stationary phase with different bonded amounts, column temperature, and sample loading capacity, on the retention, resolution, and peak shape of iopamidol were investigated. The results showed that good retention and resolution of iopamidol were realized on a C18-1 column (250 mm×4.6 mm, 10 μm) of which the bonded amount was 13.7%. Retention of iopamidol was weakened with increasing column temperature, resulting in a low resolution between iopamidol and impurities. Thus, the column temperature was adjusted to 20-30℃. Meanwhile, the increasing of loading capacities was also detrimental to retention of iopamidol or removal of impurities. Prep-HPLC was performed on the C18-1 column (270 mm×50 mm, 10 μm) with the mobile phase of water-methanol at a column temperature of 20℃. After preparation, the chromatographic purity of the iopamidol sample was 98.97% with recovery of 93.44%, and its related substances all met limited requirements. This method can reduce the impurity level effectively with a high recovery rate, which is helpful for the development of separation and purification of iopamide.