Direct profiling of endogenous biomolecules in tissue samples is considered as a promising approach to investigate metabolic-related toxicity in organisms induced by emerging pollutants. Herein, we report the application of internal extractive electrospray ionization mass spectrometry (iEESI-MS) to direct phospholipid profiling in the liver and spleen tissues of Nile tilapia exposed to perfluorooctanoic acid (PFOA). Combining positive and negative ion detection modes, 130 phospholipid signals were directly detected and identified by iEESI-MS in the tissues of Nile tilapia, including phosphatidyl cholines (PCs), sphingomyelins (SMs), phosphatidic acids (PAs), phosphatidyl ethanolamines (PEs), phosphatidyl glycerols (PGs), phosphatidyl inositols (PIs) and phosphatidyl serines (PSs). With the help of partial least squares discriminant analysis (PLS-DA) and one-way analysis of variance (ANOVA), several phospholipid signals showed a significant difference in the tissue of Nile tilapia between the control group and PFOA exposure groups. In addition, pathway analysis revealed that PFOA has a significant metabolic impact on the glycerophospholipid metabolism in Nile tilapia. Without complex sample preparation, iEESI-MS was applied to direct phospholipid profiling in the liver and spleen tissues of Nile tilapia treated with PFOA, which provided a promising methodology for investigating environmental toxicity and phospholipid-dysregulation caused by emerging pollutants in aquatic organisms.