FEN1 plays a crucial role in tumor progression and proliferation, and it has been considered a prospective cancer biomarker and druggable target. However, its detection is compromised by expenditure consumption and challenged by the lack of straightforward and high-efficiency strategies both in vitro and intracellular applications. Herein, we describe an original technique for hypersensitive detection of FEN1 by incorporating structure-specific branched DNA (BD) blocked activator with CRISPR-Cas12a methodology. Given the presence of overhanging structure at the 5′ end region of the tripartite BD, it is difficult for the BD to activate Cas12a protein which could be ascribed to the larger steric hindrance effect at the 5′ terminal regions and interferes with the approachability of the constituent elements for crRNA to fully hybridize with the complementary sequences of BD. However, the appearance of FEN1 can cleave the overhanging branch of BD to form an identifiable split DNA activator, thus successfully activating the cis/trans-cleavage activity of Cas12a. By introducing a fluorescence resonance energy transfer (FRET)-based single-stranded DNA reporter, consecutive degradation events lead to an immediately perceptible intensification in fluorescence intensity, realizing highly efficient detection of FEN1. The strategy has also been successfully applied to complex biological sample analysis and intracellular imaging, demonstrating its potential application in biochemical and molecular biology research as well as clinical diagnosis. In addition, we preliminarily verified the feasibility of integrating the established strategy with an electrochemiluminescent (ECL) platform, and confirmed that this strategy can be further expanded to other miniaturized sensing devices and has great prospects for point-of-care applications.