As a core genetic biomolecule in ecosystems, the metabolic processes of DNA, particularly DNA replication and damage repair, are regulated by Flap endonuclease 1 (FEN1). Abnormal expression and dysfunction of FEN1 may lead to genomic instability, which can induce a variety of chromosome-associated disorders, including tumours. FEN1 has emerged as a prominent tumour marker. It is crucial to precisely assess FEN1 activity and identify its associated inhibitors for both experimental research and clinical applications. However, traditional detection methods are laborious and time-consuming. Despite the increasing number of studies on biosensing strategies for detecting low-abundance FEN1 owing to the continuous innovation of new technologies and materials, they have not been systematically organised and evaluated in the public domain. Herein, we review research progress in biosensing strategies based on FEN1 detection, which are classified into nanotechnology-mediated strategies, amplification strategies, and their combinations. In addition, we summarise the existing challenges of the new methods and present a perspective on the future direction of the FEN1 analysis.