In recent years, many nucleic acid-based lateral flow assays (NALFAs) have been developed for rapid and simple detection of various analytes including DNA sequences. In a NALFA, target molecules are applied within a small volume of a rehydrating buffer. The analyte flows laterally to reach the capture molecules at where it forms a colorimetric signal. Usually, in NALFAs, capture molecules are modified for maximized adsorption on the surface. In most cases, the modification is a biotin. The biotinylated capture DNA is held at capture line by interaction with streptavidin. However, there is a demand on methods that permit utilizing unmodified capture molecules and allow a cost-effective development process. Here, we report on a biotin- and streptavidin-free model NALFA. We also present a systematic investigation on the effect of various rehydrating buffers’ composition and concentration. In addition, the impacts of a protein blocker, detergents and chaotropic and kosmotropic agents on the intensity of the signal over background were analyzed. It has been demonstrated that simultaneous presence of sodium dodecyl sulfate and bovine serum albumin doubles the intensity of visible bands in the presented unmodified NALFA. Finally, this paper presents an optimized cost-effective model system that can be adapted for hybridization-based NALFAs.