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Abstract Background Tetracycline (Tet)-regulated expression system has become a widely applied tool to control gene activity. This study aimed to improve the Tet-on system with superior regulatory characteristics. Results By comprehensively comparing factors of transactivators, Tet-responsive elements (TREs), orientations of induced expression cassette, and promoters controlling the transactivator, we developed an optimal Tet-on system with enhanced inducible efficiency and lower leakiness. With the system, we successfully performed effective inducible and reversible expression of microRNA, and presented a more precise and easily reproducible fine-tuning for confirming the target of a miRNA. Finally, the system was applied in CRISPR/Cas9-mediated knockout of nuclear factor of activated T cells-5 (NFAT5), a protective transcription factor in cellular osmoregulation. Conclusions This study established an improved Tet-on system for powerful and stringent gene regulation in functional genetic studies.