Dengue viruses are arboviruses that cause substantial human disease in tropical and subtropical regions of the world, especially in urban and semiurban areas. It is currently estimated that there are 50-100 million cases of dengue virus (DF) per annum worldwide. In Brazil, it has been recognized as an important public health problem, and an increasing number of dengue haemorrhagic fever (DHF) cases have been reported since the introduction of dengue virus type 2 (DEN-2). The dengue virus genome is a positive-sense RNA of 11kb and encodes the proteins C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5 in a single open reading frame. The protein NS3 is the second largest nonstructural protein specified by virus and is a multifunctional enzyme with serine protease activity (protease domain) as well as viral RNA replication and capping (Helicase/NTPase/RTPase domain). The aim of this work was the cloning, expression, analysis the conformational properties, and characterization of the DEN2-NS3 Helicase/NTPase activity. DEN2-NS3 (54kDa C-terminal domain) lacking the protease region was expressed in Escherichia coli (Rosetta strain) as a fusion protein in the 10x his-ubiquitin at the N-terminus. The fusion protein was isolated from the soluble bacterial extracts by nickel-agarose affinity chromatography, and after the recombinant protein was digested with yeast ubiquitin hydrolase (YUH), producing only DEN2-NS3helicase. In order to evaluate conformational changes, fluorescence experiments were performed using urea or different pH (2.3–7.9) values. The Trp residue emission spectrum clearly revealed that the concentration of 2.3M urea caused 50% denaturation. Results obtained from acidic pH did not show conformational changes, except that protein was aggregated below pH 5.5 with maximal aggregation at pH 4.4. NTPase activity was determined between pH 5.5 and 8.5 by measuring the hydrolysis of ATP to ADP and Pi. The results demonstrated that DEN2-NS3helicase protein has high activity in pH 6.5 (V₀ = 1.23 nmoles.mg^-1.minute^-1), and is 50% inhibited at pH 8.5. Theses results may be important for development of antiviral drugs aimed at the inhibition of NTP and/or RNA binding.