The article presents a novel monochrome multiplex quantitative PCR (MMQPCR) method for measuring telomere length. This method multiplexes the measurement of telomere (T) signals and single copy gene (S) signals in a single reaction, using a single fluorescent DNA-intercalating dye. The T signals are collected in early cycles before the S signals rise above baseline, while the S signals are collected at a temperature that fully melts the telomere product, ensuring accurate measurement. The correlation between T/S ratios and Terminal Restriction Fragment (TRF) lengths measured by Southern blot was stronger with this method (R² = 0.844) compared to the original singleplex method (R² = 0.677). The reproducibility of T/S results from independent runs on different days was highly significant (R² = 0.91). The method is advantageous for increasing throughput and reducing costs in epidemiological studies of telomere length. The authors also discuss the design of primers for both telomeric repeats and single copy genes, and the thermal cycling profile to ensure efficient amplification and accurate measurement. The MMQPCR method is expected to be widely applicable for studying pairs of DNA templates with different copy numbers.The article presents a novel monochrome multiplex quantitative PCR (MMQPCR) method for measuring telomere length. This method multiplexes the measurement of telomere (T) signals and single copy gene (S) signals in a single reaction, using a single fluorescent DNA-intercalating dye. The T signals are collected in early cycles before the S signals rise above baseline, while the S signals are collected at a temperature that fully melts the telomere product, ensuring accurate measurement. The correlation between T/S ratios and Terminal Restriction Fragment (TRF) lengths measured by Southern blot was stronger with this method (R² = 0.844) compared to the original singleplex method (R² = 0.677). The reproducibility of T/S results from independent runs on different days was highly significant (R² = 0.91). The method is advantageous for increasing throughput and reducing costs in epidemiological studies of telomere length. The authors also discuss the design of primers for both telomeric repeats and single copy genes, and the thermal cycling profile to ensure efficient amplification and accurate measurement. The MMQPCR method is expected to be widely applicable for studying pairs of DNA templates with different copy numbers.