Crimean-Congo hemorrhagic fever virus (CCHFV) is transmitted to humans by ticks or by direct contact with infected blood. It causes severe, often fatal, hemorrhagic diseases in humans but infection in animals is asymptomatic. CCHFV can spread from person to person and has caused many nosocomial outbreaks. Because the virus is very pathogenic for humans it must be manipulated in a biosafety level 4 (BSL4) laboratory, rendering the production of antigen for serological diagnosis difficult. To replace the native antigen, we produced a recombinant nucleoprotein expressed in mammalian cells via the recombinant Semliki Forest alphavirus replicon and developed an indirect immunofluorescence assay (IFA) as well as an enzyme-linked immunosorbent assay (ELISA) by immunocapture to detect IgM and IgG in human and animal serum. Using these methods, we analyzed clinical samples from human patients and sera from domestic animals collected in Iran and we show that this novel antigen provides a novel, sensitive and specific tool for CCHF diagnosis.