BACKGROUND: The comparative length of telomeres is considered to be related to diseases such as cancer, aging, and cardiovascular diseases. qPCR is currently one of the main methods for detecting telomere length. However, due to the unique sequence of telomeres (highly repetitive six-base sequence), it is difficult to design primers and probes to expand and detect telomere and to put internal reference gene and telomere into the same tube for detection to reduce the possible inter-pore errors and improve amplification efficiency. Besides, the stability and accuracy of the test results are greatly affected by the difference between reference genes and telomere copy number. METHODS: In this study, the single-copy genes were replaced with high-copy genes (300 copies) as the internal control to reduce the copy number difference of the internal genes and telomere. In addition, a multiplex qPCR system was constructed to detect the telomeres and an internal reference gene product. We also detected the lengths of telomeres in the genomic DNA in immortalized cells (293T and Hela) from different generations of cells. RESULTS: We detected the comparative telomere lengths of 1500 random Chinese volunteers of different ages with the multiplex qPCR method; the result shows that the comparative length of telomeres is negatively related to age. In addition, we compared our qPCR detection method with a terminal restriction fragmentation (TRF) method. Both of them were highly consistent, indicating that the qPCR method was reliable. CONCLUSIONS: In conclusion, we developed a stable, convenient, and accurate comparative telomere length detection method.