Using the DNA activator of Cas to label the secondary antibody of traditional ELISA, the CRISPR/Cas12a-based fluorescence immunoassay was presented. The sensing strategy ingeniously combines the efficient signal generation of the CRISPR/Cas12a system with antigen-antibody-specific recognition. This work provides an alternative strategy to expand the application of the CRISPR/Cas system for the detection of non-nucleic acid targets.