Cas12a ribonucleoprotein (RNP) is an RNA-guided CRISPR-associated nuclease used widely for genome editing and molecular diagnostics. Conventional detection methods rely on adopting antibody-based reagents that are expensive and lack scalability, and, moreover, only detect Cas12 enzyme rather than RNP, which is the true effector. Here, we describe a method for the rapid and quantitative detection of the effective Cas12a RNPs by the combined use of anti-CRISPR protein AcrVA1 and stem-loop RT-qPCR, achieving a limit of detection (LOD) of 1 fM in reaction buffer and 0.1 pM under biologically representative conditions.