Dengue fever as one of the most important mosquito-borne disease worldwide which approximately half of the world's population is at risk presenting several degrees of symptoms, including life-threatening dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). Dengue virus (DENV) belongs to the Flaviviridae family, and are classified into four immunologically distinct serotypes, DENV 1 to 4. Although, there is no specific treatment, early diagnosis is important for proper medical care to minimize mortality, and for prompt initiation of public health control measures. There are some diagnostic tests targeting NS1 antigen in commercial, but these methods need all the four serotypes of NS1. NS1 and NS5 are translated and appear almost simultaneously. Our work is mainly based on the fact that NS5 is the most conservative protein among all the flavivirus family, and its’ RNA dependent RNA polymerase (RdRp) domain plays a vital role in viral RNA replication. Itis a prospective biomarker for dengue infection. In this study, we constructed the DENV 2 NS5 full length and DENV2 NS5 C-terminus RdRp domain (NS5-C70) expression plasmids, a 104 kDa fragment of NS5, and a 70 kDa fragment of NS5-C70 were respectively expressed in Escherichia coli. These two purified recombinant products reacted with sera of patients infected with dengue virus in an enzyme-linked immunosorbent assay with significantly higher optical density values compared with the control sera. The recombinant DENV 2 NS5 showed strong reactivity for any of the four serotypes, while the NS5-C70 fragment showed strong reactivity for DENV 2 and 4. It is suggested DENV 2 NS5 can detect all the four serotypes patients, greatly reducing the cumbersome procedures with four viral types of antigens in early diagnostic process. The recombinant NS5 protein might be an effective antigen for dengue diagnosis, and NS5-C70 also can be used for auxiliary diagnosis.