Lateral flow assays have a great impact in diagnostics, healthcare, food and environmental analysis. This is attributed to their simplicity, ease of use, low cost and portability, in conjunction with great analytical performance. However, the low detectability, in some cases, is the main drawback of such analytical tools. Many approaches and novel nanomaterials have emerged to enhance the detectability of lateral flow assays. We have, herein, developed a new DNA-based lateral flow assay that provided a 100–1000 times signal enhancement, regarding the input of isolated DNA in the amplification reaction. Signal was improved, because all the volume of the PCR reaction was analyzed by the lateral flow assay, compared to the common assays where the volume of the analyzed PCR product is restricted down to few microliters.