Lateral flow assays (LFA) are an affordable, easy-to-use, qualitative rapid test for clinical diagnosis in nonlaboratory environments and low-resource facilities. The control line of these tests is very important to provide a valid result, confirming that the platform operates correctly. A clear, nondiffused line is desirable. The number of colored nanoparticles that reach the control line in a positive test can be very small, and they should all be trapped efficiently by the molecules adsorbed there. In this work, we proposed the use of robust biotinylated dendrimers of two different generations as signal amplifiers in control lines of LFA, able to react with streptavidin-modified gold nanoparticles. Besides the synthesis and characterization, the analytical performance as control lines will be studied, and their response will be compared with other commercially available biotinylated molecules. Finally, the utility of the dendrimer implemented in a NALF (Nucleic Acid Lateral Flow) strip was also demonstrated for detection of the amplicons obtained by double-tagging PCR (polymerase chain reaction) for the detection of E. coli as a model of foodborne pathogen.