The CRISPR/Cas12a system has been repurposed as a versatile nuclei acid bio-imaging tool, but its utility in sensing non-nucleic acid analytes in living cells has been less exploited. Herein, we demonstrated the ability of Mn2+ to accelerate cleavage kinetics of Cas12a and deployed for live-cell Mn2+ sensing by leveraging the accelerated trans-cleavage for signal reporting. In this work, we found that Mn2+ could significantly boost both the cis-cleavage and trans-cleavage activities of Cas12a. On the basis of this phenomenon, we harnessed CRISPR-Cas12a as a direct sensing system for Mn2+, which achieved robust Mn2+ detection in the concentration range of 0.5-700 μM within 15 min in complex biological samples. Furthermore, we also demonstrated the versatility of this system to sense Mn2+ in the cytoplasm of living cells. With the usage of a conditional guide RNA, this Cas12a-based sensing method was applied to study the cytotoxicity of Mn2+ in living nerve cells, offering a valuable tool to reveal the cellular response of nerve cells to Mn2+ disorder and homeostasis.