The authors describe a visual genotyping assay for the rapid and simultaneous detection of four novel variant alleles within the SIRT1 promoter region. These alleles may be considered as a risk biomarker for the onset of Parkinson disease. An amplified DNA fragment, flanking the four SIRT1 polymorphisms, is subjected to a multiplex primer extension reaction in the presence of biotin-dUTP. Extension of primers and, consequently, incorporation of the biotin-dUTP occurs only if primers are perfectly complementary to the target sequence. Detection of the extension products is achieved by a DNA biosensor that functions in a lateral-flow mode and carries multiple test spots. Each spot comprises a suspension of polystyrene microspheres functionalized with capture probes. The primer extension reaction products hybridize, through specific sequence tags, to the capture probes of the biosensor. Biotinylated extended primers are visualized by means of anti-biotin-conjugated gold nanoparticles. The genotype is simply read out by observing the formation of red test spots. The method was optimized and evaluated by analyzing three recombinant DNA fragments carrying the novel polymorphisms and 48 clinical samples from patients with sporadic Parkinson disease and from healthy individuals. The proposed genotyping method is accurate, sensitive, and cost effective. As low as 6 fmol of amplified genomic DNA was found to be sufficient to generate detectable spots for correct genotyping. The total time required for the accumulation of genotyping results takes ∼2 h.