Cytomegalovirus (CMV) is a member of the Herpesviridae family and is known to infect people of all ages. In most cases, CMV infection is asymptomatic, and the virus is cleared from the host without showing any significant symptoms. However, 1 out of 200 babies are born with congenital CMV infection, which affects multiple organs, including the brain, liver, spleen, lung, and inner ear. One long-term health problem in 1 out 5 babies born with congenital CMV infection is hearing loss. The progression of CMV-associated hearing loss in the first two years of life may lead to developmental delays in language, learning, and communication. Currently, for serological testing of CMV in patients older than 12 months, real-time polymerase chain reaction (rtPCR) is used. Although the rtPCR method quantitatively detects the CMV, this method is expensive and needs highly skilled technicians to perform the assay. Therefore, there is a need for a cost-effective, simple, and rapid diagnostic tool that can help detect CMV in newborn babies and prevent CMV-mediated pathology in pediatric as well as in future adult conditions. In recent scientific studies, gold nanoparticles (AuNP) of 100 nm, 40 nm, and 15 nm sizes have shown promising results in detecting various viruses. In our study, we developed an AuNP-based colorimetric assay for detecting CMV in pediatric-derived urine samples. For comparison purposes, the pediatric urine samples were screened through quantitative PCR (qPCR) for positive and negative CMV determination. In our assay, we explored the ability of different-sized nanoparticles to detect CMV in pediatric urine samples. Using purified CMV virions as a positive control, we have shown that AuNP can effectively detect the presence of CMV in pediatric urine samples. Therefore, this colorimetric assay may be a basis for a useful diagnostic tool for detecting CMV in newborn babies. It is a rapid and non-invasive high-throughput assay for screening for the presence of CMV at the point of care and may help provide better therapeutic outcomes for pediatric patients.