Herein, we describe a CRISPR-Cas12a sensing platform activated by a DNA ligation reaction for the sensitive detection of non-nucleic acid targets, including NAD+, ATP and polynucleotide kinase (PNK). In this design, the DNA ligation reaction triggered by these biomolecules generates DNA duplexes, which can activate the nuclease activity of Cas12a to produce amplified fluorescence signals. As a result, this work provides an alternative strategy to expand the applicability of the CRISPR-Cas system into the detection of non-nucleic acid biomolecules.