Telomerase is over-expressed in over 85% of all known human tumors. This renders the enzyme a valuable biomarker for cancer diagnosis and an important therapeutic target. The most widely used telomeric repeat amplification protocol (TRAP) assay has been questioned for telomerase detection. It is reported that human telomerase activity can be visualized by using primer-modified Au nanoparticles. The working principle is based on the elongated primers conjugated to the gold nanoparticle (AuNP) surface, which can fold into a G-quadruplex to protect the AuNPs from the aggregation. The developed simple and sensitive colorimetric assay can measure telomerase activity down to 1 HeLa cell µL−1. More importantly, this assay can be easily extended to high-throughput and automatic format. The AuNP-TS method is PCR-free and therefore avoids the amplification-related errors and becomes more reliable to evaluate telomerase activity. This assay has also been used for initial screening of telomerase inhibitors as anticancer drug agents.