High performance liquid chromatography (HPLC) on titania column was performed for phosphatidylcholine (PC) separation and analysis. To fully understand the retention mechanism of PC on titania, the effect of mobile phase type, %acetonitrile (%ACN), phosphate buffer concentration, buffer pH and flow rate were all studied. Separation depended on the eluent conditions and PC retention was attributed to the cooperation of hydrophilic interaction and ligand exchange. PC in soybean lecithin products was completely separated from other components within 6 min and quantitative analysis was achieved by an external standard method, with a correlation coefficient (R²) of 0.9947. The method precision and detection limit of PC were 3.40% and 0.2 μg, respectively, with the recovery ranging from 91% to 101%. Moreover, PC and glycerol monostearate standard mixture was successfully isolated by the proposed method, which underlined the selective affinity of titania for phosphorylated lipids. With the superiority of simplicity, rapidity and accuracy, this HPLC method based on titania is a competent method for the determination of PC in lecithin samples with prospects in the field of organo-phosphates analysis.