Cynoglossus semilaevis papillomavirus (CsPaV) is an emerging viral pathogen associated with high mortality in farmed Chinese tongue soles. A sensitive, specific, and quantitative method is needed for CsPaV surveillance and epidemiological studies. In this study, we designed primers and a probe that targeted the CsPaV L1 gene. We then established a TaqMan probe-based quantitative PCR (qPCR) assay. The assay detected as few as 5.8 × 10¹ copies/µL and was 1000-fold more sensitive than conventional PCR. The assay did not cross-react with the other aquatic pathogens tested. The intra-assay coefficients of variation ranged from 0.23% to 0.84%, and the inter-assay coefficients ranged from 0.56% to 1.48%. These results showed that the assay had good repeatability. We used the assay to study the tissue distribution and temporal dynamics of CsPaV after experimental infection. Healthy Chinese tongue soles received an intraperitoneal injection of 300 µL of inoculum containing 3.66 × 10⁵ CsPaV copies/µL. Three fish were sampled at 1, 2, 3, 5, and 7 days post-infection. CsPaV DNA was detected in all tissues examined. The kidney had the highest viral DNA load, which reached 2.47 × 10⁷ copies/mg at 7 days post-infection. Among 176 clinically diseased Chinese tongue soles collected between 2023 and 2026, 80.68% tested positive for CsPaV. CsPaV DNA was also detected in several other aquatic species. These findings provide a sensitive tool for CsPaV surveillance and molecular epidemiological studies. The assay may also support disease prevention and control in Chinese tongue sole aquaculture.