Human gnathostomiasis is an emerging food-borne parasitic disease caused by nematodes of the genus Gnathostoma. Currently, serological tests are commonly applied to support clinical diagnosis. In the present study, a simple and rapid filtration-based test, dot immune-gold filtration assay (DIGFA) was developed using a partially purified antigen of Gnathostoma third-stage larvae (L3). A total of 180 serum samples were tested to evaluate the diagnostic potential of DIGFA for gnathostomiasis. The diagnostic sensitivity and specificity were 96.7% (29/30) and 100% (25/25), respectively. The cross-reactivity with sera from other helminthiasis patients ranged from 0 to 4%, with an average of 1.6% (2/125). DIGFA using a partially purified L3 antigen was not only simple and rapid, but also more accurate than standard assays for the diagnosis of human gnathostomiasis. DIGFA may represent a promising tool for application in laboratories or in the field, without requiring any instrumentation.