Tuberculosis　is　one　of　the　most　serious　infectious　diseases,　resulting　in　death　worldwide.　Traditional　detection　methods　are　not　enough　to　meet　the　clinical　requirements　of　rapid　diagnosis,　high　specificity,　and　high　sensitivity.　Fast,　sensitive,　and　accurate　detection　of　Mycobacterium　tuberculosis　(MTB)　is　urgently　needed　to　treat　and　control　tuberculosis　disease.　Clustered　regularly　interspaced　short　palindromic　repeats　(CRISPR)-associated　proteins　(Cas12a)　exhibit　strong　nonspecific　degradation　ability　of　exogenous　single-strand　nucleic　acids　(trans　cleavage)　after　specific　recognition　of　target　sequence.　We　purified　Cas12a　protein　and　selected　a　proper　guide　RNA　based　on　conserved　sequences　of　MTB　from　designed　guide　RNA　library.　Then,　we　proposed　a　novel　detection　method　based　on　recombinase　polymerase　amplification　and　CRISPR/Cas12a　nuclease　system　for　specific　and　sensitive　detection　of　MTB　DNA.　The　assay,　based　on　fluorescence　detection,　showed　4.48　fmol/L　of　limit　of　detection　and　good　linear　correlation　of　concentration　with　fluorescence　value　(R-2　=　0.9775).　It　also　showed　good　performance　in　distinguishing　other　bacteria.　Furthermore,　its　clinical　performance　was　evaluated　by　193　samples　and　showed　sensitivity　of　99.29%　(139/140)　and　specificity　of　100%　(53/53)　at　99%　CI,　compared　with　culture　method.　Taken　together,　the　CRISPR/Cas12a　system　showed　good　specificity,　excellent　sensitivity,　and　excellent　accuracy　for　MTB　detection,　and　it　meets　requirements　of　MTB　detection　in　clinical　samples　and　has　great　potential　for　clinical　translation.