Simple SummaryDengue infection, a global threat, necessitates prompt action. An early diagnosis based on the dengue antigenic target is preferable. The current method of dengue diagnosis is based on antibodies, which have many disadvantages such as expensive production process and non-uniform batch synthesis. Aptamers are able to address shortcomings of antibodies, and with this in mind, we set out to isolate RNA aptamers against the NS1 protein of dengue virus serotype 2. In this study, we isolated two aptamers known as DENV-3 and DENV-6. Upon truncation to TDENV-3 and TDENV-6a, their performance in the direct ELASA improved. Using these aptamers, a sandwich ELASA was successfully developed for the detection of dengue NS1. The sensitivity of the sandwich ELASA was further improved by stabilization of the truncated aptamers and our repeated incubation strategy, attaining a LOD of 2 nM using NS1 spiked into human serum prepared using a dilution factor of 1:2000.AbstractDengue infection, caused by the dengue virus, is a global threat which requires immediate attention and appropriate disease management. The current diagnosis of dengue infection is largely based on viral isolation, RT-PCR and serology-based detection, which are time-consuming and expensive, and require trained personnel. For early diagnosis of dengue, the direct detection of a dengue antigenic target is efficacious, and one such target is NS1. NS1-based detection is primarily antibody-centric and is beset by drawbacks pertaining to antibodies such as the high cost of synthesis and large batch-to-batch variation. Aptamers are potential surrogates of antibodies and are much cheaper, without exhibiting batch-to-batch variation. Given these advantages, we sought to isolate RNA aptamers against the NS1 protein of dengue virus serotype 2. A total of 11 cycles of SELEX were carried out, resulting in two potent aptamers, DENV-3 and DENV-6, with dissociation constant values estimated at 37.57 ± 10.34 nM and 41.40 ± 9.29 nM, respectively. These aptamers can be further miniaturized to TDENV-3 and TDENV-6a with an increased LOD upon their usage in direct ELASA. Moreover, these truncated aptamers are highly specific against the dengue NS1 while showing no cross-reactivity against the NS1 of the Zika virus, the E2 protein of the Chikungunya virus or the LipL32 protein of Leptospira, with target selectivity retained even in human serum. The usage of TDENV-3 as the capturing probe and TDENV-6a as the detection probe underpinned the development of an aptamer-based sandwich ELASA for the detection of dengue NS1. The sensitivity of the sandwich ELASA was further improved with the stabilization of the truncated aptamers and the repeated incubation strategy, which enabled a LOD of 2 nM when used with the target NS1 spiked in human serum diluted at 1:2000.