The viral genome titer is a crucial indicator for the clinical dosing, manufacturing, and analytical testing of recombinant adeno-associated virus (rAAV) gene therapy products. Although quantitative PCR and digital PCR are the common methods used for quantifying the rAAV genome titer, they are limited by inadequate accuracy and robustness. The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a biosensor is being increasingly used in virus detection; however, there is currently no report on its application in the titer determination of gene therapy products. In the present study, an amplification-free CRISPR-Cas12a assay was developed, optimized, and applied for rAAV genome titer determination. The assay demonstrated high precision and accuracy within the detection range of 4 × 10 9 and 10 11 vg/mL. No significant difference was observed between the Cas12a and qPCR assay results ( p < 0.05, t test). Moreover, Cas12a exhibited similar activity on both single-stranded and double-stranded DNA substrates. Based on this characteristic, the titers of positive-sense and negative-sense strands were determined separately, which revealed a significant difference between their titers for an in-house reference AAV5-IN. This study presents the inaugural report of a Cas12a assay developed for the titer determination and composition analysis of the rAAV genome.