With hydrophilic surface and high density of functional groups, MXene can efficiently adsorb single-stranded DNA to enhance target-induced strand release and quench the fluorescence. Herein, MXene is coupled with CRISPR-Cas12a to sensitively detect LPS and bacteria. Specifically, the aptamer is well designed to initiate the trans-cleavage activity of CRISPR-Cas12a to indiscriminately cleave single-stranded DNA, resulting it to be far away from MXene and the recovery of fluorescence. The target can effectually induce the release of the aptamer strand from the hybrid duplex with the assistance of MXene. The formed aptamer/target complex will inhibit the activation of CRISPR-Cas12a and its trans-cleavage on single-stranded DNA. The established method can selectively and sensitively quantify LPS and Gram-negative bacteria in different samples with detection limits of 11 pg/mL and 23 CFU/mL, respectively. Our study provides a new insight for exploration of universal analytical methods based on MXene coupled with CRISPR-Cas12a.