Osteosarcoma is the most frequent primary malignant bone tumor, composed of mesenchymal cells producing osteoid and immature bone. The sensitive detection of telomerase plays a pivotal role in the early diagnosis and therapeutic treatment of osteosarcoma. We report here an in vitro strategy for sensitive telomerase activity detection through the integration of rolling circle amplification (RCA) and a clustered regularly spaced short palindrome repeats (CRISPR)-Cas12a system. In the proposed strategy, telomerase substrate (TS) primers are easily controlled to extend five bases (GGGTT) to give short telomerase extension products (TEP) with definite lengths without adding dATP. The resulting short TEPs can then cyclize the padlock through hybridizing with its two terminals and thus initiate the following RCA. To obtain an improved sensitivity, the CRISPR-Cas12a system is attached to collaterally cut surrounding DNA reporter probes after recognizing the target single strand DNA sequence in the RCA products. The highlights of this strategy are as follows: (i) the short TEP triggered strategy is excellent at detecting low telomerase activity and thus contributes to the early diagnosis of malignant tumors; (ii) highly sensitive telomerase activity detection which is easy to operate from RCA initiated CRISPR-Cas12a; (iii) opening up of a new avenue for telomerase activity detection with a CRISPR-Cas12a system. Finally, the proposed strategy exhibited sensitive telomerase activity detection under optimized experimental parameters and has great application potential for the clinical diagnosis of malignant tumors and the development of anti-cancer drugs.