Abstract: We applied digital PCR targeting mitochondrial DNA (mtDNA) and nuclear DNA (nuDNA) markers to investigate environmental DNA (eDNA) dynamics during artificial propagation of the critically endangered Yangtze sturgeon (Acipenser dabryanus). Species-specific primers and TaqMan probes were designed to quantify mtDNA and nuDNA concentrations and their ratio (nuDNA/mtDNA) across different reproductive stages. Both mtDNA and nuDNA levels increased rapidly and reached their peaks after spawning. In the male pond, concentrations rose to 27-fold (mtDNA) and 130-fold (nuDNA) of pre-spawning levels following sperm release; in the female pond, concentrations increased to 8-fold (mtDNA) and 13-fold (nuDNA) after ovulation. The nuDNA/mtDNA ratio peaked at 0.14 and 0.09 in the male and female ponds, respectively. Overall, peak mtDNA concentrations were approximately 11—12 times higher than those of nuDNA and remained detectable for 24—36h, indicating greater persistence. In contrast, nuDNA concentrations declined sharply within 12h post-spawning, demonstrating high temporal sensitivity to spawning events. Our findings demonstrate that eDNA monitoring effectively captures molecular signatures and temporal patterns of A. dabryanus reproductive activity, offering a robust, non-invasive tool for studying the reproductive ecology and informing conservation strategies for endangered fish species.