CRISPR-Cas12a system has been widely utilized for genome editing and molecular diagnostics applications. However, existing CRISPR-Cas12a systems still have much room for improving its functionality to expand their applications. To achieve this goal, a thorough understanding of the conformational dynamics of CRISPR-Cas12a system is essentially required. In this regard, single-molecule fluorescence assays have been recently applied to study the dynamics of CRISPR-Cas12a. In this review, we introduce single-molecule fluorescence studies on CRISPR-Cas12a system and provide practical guidelines required for reliable implementation of the single-molecule fluorescence methods to study the molecular mechanism of DNA cleavage reaction of CRISPR-Cas12a system.