Anisakiasis is an infectious parasitic disease contracted by eating third stage larvae of Anisakis simplex (L3) carried by marine fishes. Human anisakiasis was researched for specific IgG with L3 excretory secretory products (ESP). L3ESP were prepared by daily harvesting of culture supernatant from day 2 to day 5, using two kinds culture media of RPMI-1640 and phosphate buffered saline (PBS). When the sera from persons diagnosed with anisakiasis by means of endoscopy were analyzed using indirect ELISA and Western blot, the sera was classified into four groups depending on specific antigen recognition patterns. In addition, the presence of a new antigen for L3, located at less than 13 kDa (AgLT13) was demonstrated in L3ESP with a modified Western blot condition. The production of AgLT13 was mainly found in L3ESP harvested both on day 2 and day 3, and that in PBS was predominant over that in RPMI-1640. Among those sera, the high reactive sera to L3ESP-day two prepared with phosphate buffer in indirect ELISA recognized AgLT13, and 33% of the low reactive sera did so. These results indicate that the binding pattern of human L3 specific antibody is diverse depending on L3ESP preparations and that AgLT13 demonstrated with a Western blot condition is a specific antigen for L3.