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　&　PRIME;-flap　of　double-flap　DNA　substrate　modified　on　SAMBs　was　firstly　cleaved　by　FEN1.　Large　amount　of　cleaved　5　&　PRIME;-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　x　10-3　to　3.1　x　10-2　U　&　mu;L-1　with　a　LOD　of　1.6　x　10-3　U　&　mu;L-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　x　10-3-6.1　x　10-2　U　&　mu;L-1　with　a　LOD　of　1.1　x　10-3　U　&　mu;L-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.