Dengue virus (DENV) is a mosquito-borne flavivirus and a major international public health concern in many tropical and sub-tropical areas worldwide. DENV is divided into four major serotypes, and infection with one serotype leads to immunity against the same, but not the other serotypes. The specific diagnosis of DENV-infections via antibody-detection is problematic due to the high degree of cross-reactivity displayed by antibodies against related flaviviruses, such as West Nile virus (WNV), Yellow Fever virus (YFV) or Tick-borne encephalitis virus (TBEV). Especially in areas where several flaviviruses co-circulate or in the context of vaccination e.g. against YFV or TBEV, this severely complicates diagnosis and surveillance. Most flavivirus cross-reactive antibodies are produced against the highly conserved fusion loop (FL) domain in the viral envelope (E) protein. We generated insect-cell derived recombinant E-proteins of the four DENV-serotypes which contain point mutations in the FL domain. By using specific mixtures of these mutant antigens, cross-reactivity against heterologous flaviviruses was strongly reduced, enabling sensitive and specific diagnosis of the DENV-infected serum samples in IgG and IgM-measurements. These results have indications for the development of serological DENV-tests with improved specificity.