Phospholipids and their metabolites play an important role in a variety of cellular processes including cell-cell adhesion, cell growth and differentiation, apoptosis, phagocytosis as well as storage of energy. In this study, the phospholipid composition of cancer tissue and adjacent normal tissue from humans and animals were analyzed by internal extractive electrospray ionization mass spectrometry (iEESI-MS). Extractive solvent at high voltage (+5.5 kV) was injected into tissue samples using a fused silica capillary at a flow rate of 0.5–1.0 μL min⁻¹, producing fine charged droplets containing analytes of tissue samples at the tip of the sample. Charged droplets were directly sampled to the atmospheric inlet of a mass spectrometer. Out of 21 different ratios of CH₃OH-H₂O solvent mixture, the ratio CH₃OH-H₂O (30:70,V/V) showed the optimal phospholipids extraction and visibility in MS. A large number of phospholipids from different tissue samples (such as cancer tissue and adjacent normal tissue of lung cancer, esophageal tissue, pork, beef, porcine heart and porcine lung) were obtained simultaneously by iEESI-MS analysis. The experimental results demonstrated that iEESI-MS was characterized by minimal sample pretreatment, low sample consumption, and rapid analysis (the analysis time per sample was less than 1 min), and the selectivity and sensitivity of iEESI-MS could be improved by choosing proper solvent. Importantly, the experimental results provided new information for further studies of phospholipids in biological tissues.