We have prepared a chemically anchored monolayer of PEG (poly(ethylene glycol)) and phospholipid mixture (PEG/phospholipid) on a methacryloyl-terminated substrate by in situ photopolymerization. Both monoacryloyl phospholipid (acryloyl-PC, 1-palmitoyl-2-[12-(acryloyloxy)dodecanoyl]-sn-glycero-3-phosphocholine) and monoacryloyl PEG (acryloyl-PEG, 12-(acryloyloxy)dodecanoyl-PEG) were synthesized by modifying phospholipid and PEG with 12-(acryloyloxy)-1-dodecanoic acid and 12-(acryloyloxy)-1-dodecanol, respectively. The surface pressure-area (Pi-A) isotherm showed that acryloyl-PEG molecules were stable in the phospholipid monolayer and that they could be evenly inserted into a phospholipid monolayer at the air/water interface. By adding 10 mol % acryloyl-PEG into phosholipid vesicles, we could produce a PEG/phosholipid monolayer on methacryloyl-terminated substrates using vesicle fusion for 3 h. Then, this polymerizable PEG/phospholipid monolayer was in situ photopolymerized onto a methacryloyl-terminated substrate with eosin Y/triethanolamine as co-initiators. Optimal vesicle fusion and irradiation condition were determined with respect to the vesicle fusion time and duration of irradiation. As confirmed by atomic force microscopy and X-ray reflectivity studies, the polymerized PEG/phosholipid surface formed a PEG-covered phospholipid monolayer with thicknesses of 3 and 6 nm for the base phospholipid monolayer and the covering PEG layer, respectively. The chemical anchoring efficiency of polymerized PEG and phospholipid molecules, which was calculated by the relative carbon ratio of each surface before and after methanol washing using X-ray photoelectron spectroscopy, was 98%. This polymerized PEG/phosholipid monolayer showed good stability in organic solution due to firm chemical anchoring to a solid surface.