For the diagnosis of human fasciolosis, a new dipstick assay was developed and its diagnostic efficacy was evaluated in comparison to enzyme linked immunosorbent assay (ELISA) using Fasciola gigantica excretory-secretory antigen. Sera samples were collected from patients with confirmed fasciolosis and patients with other parasitic diseases as well as from normal healthy individuals. The sensitivity, specificity, and diagnostic accuracy of the dipstick assay as well as ELISA were 100%, 96.7%, and 98.3%, respectively. The dipstick assay is extremely easy to apply, rapid (15 min), with a visually interpreted result, field portable, inexpensive, reagent conservative, and as being both sensitive and specific in diagnosis of human fasciolosis; the test could be an acceptable alternative for use in clinical laboratories lacking specialized equipment needed for ELISA.