A DNA micro-optode for dengue virus detection was developed based on the sandwich hybridization strategy of DNAs on succinimide-functionalized poly(n-butyl acrylate) (poly(nBA-NAS)) microspheres. Gold nanoparticles (AuNPs) with an average diameter of ~20 nm were synthesized using a centrifugation-based method and adsorbed on the submicrometer-sized polyelectrolyte-coated poly(styrene-co-acrylic acid) (PSA) latex particles via an electrostatic method. The AuNP-latex spheres were attached to the thiolated reporter probe (rDNA) by Au-thiol binding to functionalize as an optical gold-latex-rDNA label. The one-step sandwich hybridization recognition involved a pair of a DNA probe, i.e., capture probe (pDNA), and AuNP-PSA reporter label that flanked the target DNA (complementary DNA (cDNA)). The concentration of dengue virus cDNA was optically transduced by immobilized AuNP-PSA-rDNA conjugates as the DNA micro-optode exhibited a violet hue upon the DNA sandwich hybridization reaction, which could be monitored by a fiber-optic reflectance spectrophotometer at 637 nm. The optical genosensor showed a linear reflectance response over a wide cDNA concentration range from 1.0 × 10-21 M to 1.0 × 10-12 M cDNA (R2 = 0.9807) with a limit of detection (LOD) of 1 × 10-29 M. The DNA biosensor was reusable for three consecutive applications after regeneration with mild sodium hydroxide. The sandwich-type optical biosensor was well validated with a molecular reverse transcription polymerase chain reaction (RT-PCR) technique for screening of dengue virus in clinical samples, e.g., serum, urine, and saliva from dengue virus-infected patients under informed consent.