A colorimetric and photothermal dual readout biosensor for Flap endonuclease 1 (FEN1) quantification was developed on the basis of target-prevented gold nanoparticles (AuNPs) aggregation. The exposed 5′-flap of double-flap DNA substrate modified on SAMBs was firstly cleaved by FEN1. Large amount of cleaved 5′-flap remained in the supernatant after simple magnetic separation, which can adsorb on the surface of AuNPs and effectively prevent the dispersed AuNPs from aggregation under high ionic concentration, accompanied with the color changing of the system, which can be recognized by nake eyes easily. The absorption intensity at 528 nm shows a good linear relationship with the increasing FEN1 concentration from 5.0 × 10-3 to 3.1 × 10-2 U μL-1 with a LOD of 1.6 × 10-3 U μL-1 (S/N = 3). Given the aggregated AuNPs have higher photothermal effect than that of the dispersed AuNPs, the target-prevented AuNPs aggregation avoids a sharp increase of temperature for the system under the laser radiation. The temperature change is linearly correlated with the FEN1 concentration in the range of 3.1 × 10-3–6.1 × 10-2 U μL-1 with a LOD of 1.1 × 10-3 U μL-1. The whole detection process can be completed within 1 h. The proposed system had been applied to detect FEN1 concentration in serum samples with satisfied results, which can be applied in resource-limited area easily and quickly.