The study aimed to explore the antigenic proteins in third-stage Anisakis spp. larvae from mackerel tuna. This research used 300 Anisakis spp. larvae from 110 mackerel tuna fish from Brondong, Lamongan, East Java, Indonesia. The research was carried out in several stages: isolation and identification of Anisakis spp.; isolation of soluble proteins and measurement by spectrophotometry; whole-protein characterization with SDS-PAGE; rabbit immunization for polyclonal antibody production; antibody titer measurement with indirect ELISA; and antigenic protein characterization via Western blot. Spectrophotometry results of the larvae showed homogenate protein level of 6833.3 μg/ml. Protein analysis of whole worm extract of the larvae using SDS-PAGE yielded 21 bands, with molecular weights between 12 kDa-238 kDa. Further, protein characterization via Western blot produced six antigenic protein bands: 64 kDa, 45 kDa, 38 kDa, 30 kDa, 27 kDa and 23 kDa. In conclusion, Anisakis spp. larvae in mackerel tuna contain six types of antigenic proteins, which might play a role in hypersensitivity or allergic reactions.