Pterygia, caused by fibrovascular growth of conjunctiva, are a common ophthalmic disease. However, the molecular composition of pterygium tissue has not been completely understood, and therefore the aim of this study is to investigate the spectroscopic differences between normal human bulbar conjunctiva and human pterygium tissue using a confocal Raman system. The high signal-to-noise ratio spectra from pterygium and conjunctiva tissue were obtained by this technique without any sample preparation and the time of detection required less than 3 min. Comparing Raman spectra of two types of tissue, there are obvious changes, including intensity decrease at ~1585 cm–1 and intensity increase at ~1748, 1156, and 1521 cm–1 with the lesion of conjunctiva. Additionally, the amide I vibrational mode of proteins in conjunctiva is significantly different than that in pterygium. The use of pathology, immunology, and the peroxidation of the lipids in conjunction with the Raman results indicate that the presence of additional elastic fibers, mast cells, and lymphocytes in pterygium, as compared with normal bulbar conjunctiva, have fewer unsaturated fat acids. The present study demonstrates that Raman spectroscopy can be potentially applied to diagnose pterygia clinically.