Nucleic acid amplification strategies have successfully dominated ultrasensitive bioassays, but they sometimes bring high time-consumption, multi-step operation, increased contamination risk, and mismatch-related inaccuracy. We proposed a nucleic acid amplification-free method called the AuNPs-tagging based CRISPR-Cas12a bioassay platform. The signal amplification was realized by integrating the self-amplification effect of CRISPR-Cas12a with the enhancement effect of the large number of detectable atoms inside each gold nanoparticle. The proposed method achieved a low LOD of 1.05 amol in 40 min for HIV-related DNA.