The diagnostic values of immunoglobulin G subclass antibodies from patients with gnathostomiasis were assessed by immunoblot technique. Antigen was prepared from crude extracts of Gnathostoma spinigerum advanced third-stage larvae obtained from naturally infected eels. The sera were obtained from 14 parasite-confirmed gnathostomiasis cases, 63 patients with other helminthic infections and 13 healthy controls. Nine prominent IgG4 reactive bands appeared with molecular weights of 94, 51, 47, 43, 38, 24, 21, 20 and 15 kDa. The diagnostic sensitivity of each of the nine reactive bands ranged from 100% to 64.3% in 14 parasite-confirmed gnathostomiasis cases. All (100%) confirmed cases recognized the 21 kDa antigenic band, but not other helminthic infections or parasite-free control. Recognition of 21 kDa antigen in G. spinigerum advanced third-stage larvae crude extracts is the most specific diagnostic marker for human gnathostomiasis, with 100% sensitivity and specificity. The 20 and 24 kDa protein bands were additional diagnostic bands for confirming diagnosis of infection where the 21 kDa band was faint. No specific binding of IgG1, IgG2, or IgG3 antibodies was observed in any sera from confirmed gnathostomiasis cases.