Continued development of clustered regularly interspaced short palindromic repeats (CRISPR)-powered biosensing system on the electrochemical interface is vital for accurate and timely diagnosis in clinical practice. Herein, an electrochemical biosensor based on manganese metal-organic frameworks (MOFs)-enhanced CRISPR (MME-CRISPR) is proposed that enables the efficient detection of circulating tumor DNA (ctDNA). In this design, customized enzyme stimulants (Mn) are co-assembled with Cas12a/crRNA to form enzyme-MOF composites, which can be released quickly under mild conditions. The MOFs-induced proximity effect can continuously provide adequate Mn to sufficiently interact with Cas12a/crRNA during the release process, enhancing the trans-cleavage activity of complex available for biosensor construction. The MOFs-based enzyme biocomposites also afford efficient protection against various external stimulus. It is demonstrated that the developed biosensor can achieve ultrasensitive detection of epidermal growth factor receptor L858R mutation in ctDNA with a low detection limit of 0.28 fm without pre-amplification. Furthermore, the engineered mismatch crRNA enables the biosensor based on MME-CRISPR to detect single nucleotide variant with a high signal-to-noise ratio. More importantly, it has been successfully used to detect the targets in clinical practice, requiring low-dose samples and a short time. This strategy is believed to shed new light on the applications of cancer diagnosis, treatment, and surveillance.