To characterize the ultraviolet (UV) light-absorbing function of the ascidian tunic, the light absorption spectrum was compared in 22 ascidian species collected from tropical and temperate waters. Non-photosymbiotic ascidians (17 species) had transparent tunics transmitting both visible and UV light, or pigmented or opaque tunics equally absorb both UV and visible light. However, a prominent absorption peak around 320 nm was exclusively found in the colonial ascidians hosting the algal symbiont Prochloron sp., and this absorption peak corresponded to absorption of UV-A (320–400 nm) and UV-B (280–320 nm). The UV-absorbing substances were extracted with methanol. They were mycosporine-like amino acids (MAAs): mycosporine-glycine, palythine, shinorine, and porphyra-334. The MAAs are thought to be synthesized by the shikimic acid pathway that does not occur in animals. Since the isolated Prochloron cells contain MAAs, the symbionts are the most possible candidates as the source of the MAAs in the tunic. In Diplosoma virens, the composition of MAAs was different between isolated Prochloron cells and colony residue from which Prochloron cells were extracted.