The secreted form of the dengue virus (DENV) nonstructural-1 (NS1) glycoprotein has been shown to be useful for the diagnosis of DENV infections in patients’ serum samples. In a number of studies, the sensitivity of the commercially available DENV NS1 glycoprotein detection assays was higher against some DENV serotypes (DENV-1 > DENV-3 > DENV-2 = DENV-4) than others and were also lower using patients’ serum samples with secondary versus primary DENV infections. In this study, 471 DENV-4 positive acute phase patients’ serum samples were selected from a large panel collected in Brazil from March 2011 to October 2012 by RT-PCR and/or virus isolation followed by serotype determination. The sera from primary (n = 228) and secondary (n = 238) DENV-4 infections were identified using IgM and IgG capture ELISAs. The sensitivity of a commercial DENV NS1 glycoprotein detection ELISA was then assessed when these serum samples were not pre-treated or pre-treated by acid or heat dissociation prior to being tested. Acid and heat dissociation of patients’ serum samples with primary and secondary DENV-4 infections increased significantly the sensitivity of the DENV NS1 glycoprotein detection ELISA from 54.4% to 77.2% (p < 0.05) and 82% (p < 0.05) and from 39.1% to 63.9% (p < 0.05) and 73.1% (p < 0.05), respectively. Treatment of DENV infected patients’ serum samples using simple and rapid heat dissociation step (100 ◦C for 5 min) was, therefore, shown to be very useful for increasing the sensitivity of the DENV NS1 glycoprotein detection ELISA using serum samples from either primary or secondary DENV infected patients.