Abstract Objective To develop a gold nanoparticle (AuNP) assay for direct detection of unamplified HURP RNA in urine. Design and methods HURP RNA was extracted from urine samples (50 bladder carcinoma patients, 25 benign bladder lesions, and 25 controls) and further purified using magnetic nanoparticles (MNPs), functionalized with HURP RNA-specific oligonucleotides, and then detected by RT-PCR or gold nanoparticles. Results The developed HURP RNA AuNP assay has a sensitivity and a specificity of 88.5% and 94%, respectively, and a detection limit of 2.4nmol/L. The concordance between the HURP AuNP assay with RT-PCR after RNA purification using functionalized MNPs was 97%. Conclusions The developed colorimetric HURP RNA AuNP assay is sensitive, simple, and can aid noninvasive diagnosis of bladder cancer.