Objective The present study attempted to design novel miRNA expression cassettes (MEC) targeting specific consensus sequences of hTERT and hTR, which remedied the degradability and cytotoxicity and provided a convenient method for miRNA synthesis.Methods The MECs specific to hTERT and hTR were constructed by overlap polymerase chain reaction (PCR). Telomeric repeat amplification protocol (TRAP)-silver staining and TRAP real-time PCR analysis were used to determine the telomerase activity. The telomere length was determined by real-time PCR, whereas cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cell apoptosis rate and cell cycle were assessed using the annexin V/propidium iodide (PI) double staining and PI single staining assays, respectively, coupled with flow cytometry.Results The telomerase-specific MECs were successfully constructed. Each MEC inhibited the telomerase activity differently. Telomerase silencing could induce immediate growth arrest in the G0/G1 phase and led to retinoblastoma (RB) cell apoptosis.Conclusion miRNA-mediated telomerase silencing is an efficient strategy to impair RB cell growth. A robust system must be developed to fully explore the efficacy of miRNAs. The constructed MECs exhibited a strong RNAi effect and thus may be utilized to effectively screen RNAi-targeted sequences for RB gene therapy.