Human telomerase is a specialized ribonucleoprotein polymerase and is used as a clinical cancer marker and special target for chemotherapy. Traditional telomerase detection uses “telomerase repeat amplification protocol” (TRAP) assay. However, TRAP is questioned because it requires use of DNA polymerases, which is susceptible to polymerase chain reaction (PCR)-derived artifacts and time-consuming. Here, a novel PCR-free, electrocatalytic assay for signal-amplified detection of telomerase activity using graphene-mesoporous silica-dispersed palladium nanoparticles-based probe carrier platform to improve probe-target recognition is reported. The low background noise is achieved by using DNA site-specific cleavage endonuclease and the detection signal is amplified by using hemoglobin. As a highly efficient electron sink, hemoglobin does not carry out direct reduction at the surface, minimizing false positives. These merits make this assay highly sensitive, achieving the sensitivity comparable to TRAP. The developed electrocatalysis assay is PCR-free, simple in design, and fast in operation, therefore avoiding PCR amplification-related errors, which make it more reliable to evaluate telomerase activity for clinical use.