The visual detection of a disease biomarker is a promising strategy due to its simplicity and low cost, but the complexity of biological samples limits its application. Herein, a reliable and ultrasensitive colorimetric assay was proposed for detecting telomerase activity in crude cancer cell extracts. A telomerase substrate (TS) primer was immobilized onto magnetic beads (MBs) to form a MB/TS complex. In the presence of telomerase and deoxyribonucleotide triphosphates (dNTPs), the TS primer was elongated by adding multiple telomeric repeats (TTAGGG)_n to the 3′ end of the TS. The telomeric repeats of the elongated TS (ETS) hybridized with its short complementary DNA (cDNA) to specifically capture peroxidase onto MBs. After magnetic separation, the activity of telomerase was detected by monitoring the change in the color or absorbance of the peroxidase-catalyzed H₂O₂/TMB reaction. Magnetic separation greatly eliminated the non-specific interference to ensure reliability and improve the signal-to-noise (S/N) ratio. The mean telomerase activity equivalent to 5 HeLa cells and 1 HeLa cell was reliably detected with the naked eye and UV-vis spectroscopy, respectively. More importantly, the telomerase activity of 5 and 20 HeLa cells was detected via UV-vis spectroscopy and the naked eye, respectively. The differences in the telomerase activity of four carcinoma cell lines and one normal cell line were discriminated visually. Even more strikingly, the telomerase activity of 10 and 50 HeLa cells in human serum was detected by change in the absorbance and color of the TMB/H₂O₂ solution, respectively. Therefore, it offers an ultrasensitive and reliable colorimetric assay for the visual detection of telomerase activity, which holds promising potential to detect telomerase activity in a complex pathological sample.