Flap endonuclease 1 (FEN1) is a specific enzyme capable of recognizing and cleaving triplex DNA structures and releasing 5'-flap fragments. It plays a crucial role in the DNA metabolism of cells, participating in DNA replication and the repair of damaged DNA. Additionally, FEN1 is overexpressed in various tumor tissues, promoting tumor progression and drug resistance through different regulatory mechanisms. However, few significant advancements have been made in sensitive analytical methods for detecting FEN1. Herein, in this study, we present a highly sensitive flow cytometry biosensor with solid-phase interface-mediated primer exchange reaction amplification (FCsperA) for FEN1 analysis. By comparing homogeneous PER amplification (h-PER), we found that solid-phase interface-mediated PER amplification (s-PER) effectively suppressed background signals, leading to a higher signal-to-background (S/B) ratio exceeding ∼46-fold when FEN1 was at 1 × 10-3 U/μL. Combining the high efficiency of s-PER, the strong suppression of background signals, and the highly precise flow cytometry assay, FCsperA showed high sensitivity, with a limit of detection (LOD) for FEN1 of 2.53 × 10-6 U/μL. Notably, FCsperA exhibited high selectivity and exceptional anti-interference ability, making it applicable for detecting FEN1 in cells and serum samples. The outstanding performance of FCsperA allowed for sensing FEN1 in ∼10 cancer cells. Additionally, FCsperA demonstrated the potential for screening inhibitors of FEN1.