Telomerase　is　one　of　the　most　common　markers　of　human　malignant　tumors,　such　as　uterine,　stomach,　esophageal,　breast,　colorectal,　laryngeal　squamous　cell,　thyroid,　bladder,　and　so　on.　It　is　necessary　to　develop　some　sensitive　but　convenient　detection　methods　for　telomerase　activity　determination.　In　this　study,　a　label-free　and　ultrasensitive　electrochemiluminescence　(ECL)　biosensor　has　been　fabricated　to　detect　the　activity　of　telomerase　extracted　from　HeLa　cells.　Thiolated　telomerase　substrate　(TS)　primer　was　immobilized　on　the　gold　electrode　surface　through　gold-sulfur　(Au-S)　interaction　and　then　elongated　by　telomerase　specifically.　Then,　it　was　hybridized　with　complementary　DNA　to　form　double-stranded　DNA　(dsDNA)　fragments　on　the　electrode　surface,　and　Ru(phen)(3)　(2+)　has　been　intercalated　into　the　dsDNA　grooves　to　act　as　the　ECL　probe.　The　enhanced　ECL　intensity　has　a　linear　relationship　with　the　number　of　HeLa　cells　in　the　range　of　5　similar　to　5000　and　with　a　detection　limit　of　2　HeLa　cells.　The　proposed　ECL　biosensor　has　high　specificity　to　telomerase　in　the　presence　of　common　interferents.　The　relative　standard　deviations　(RSDs)　were　＜　5　%　at　100　HeLa　cells.　The　proposed　method　provides　a　convenient　approach　for　telomerase-related　cancer　screening　or　diagnosis.