An　ultrasensitive　electrochemical　biosensor　for　detecting　p53　gene　was　fabricated　based　on　heated　gold　disk　electrode　coupling　with　endonuclease　Nt.BstNBI-assisted　target　recycle　amplification　and　alkaline　phosphatase　(ALP)-based　electrocatalytic　signal　amplification.　For　biosensor　assembling,　biotinylated　ssDNA　capture　probes　were　first　immobilized　on　heated　Au　disk　electrode　(HAuDE),　then　combined　with　streptavidin-alkaline　phosphatase　(SA-ALP)　by　biotin-SA　interaction.　ALP　could　catalyze　the　hydrolysis　of　ascorbic　acid　2-phosphate　(AAP)　to　produce　ascorbic　acid　(AA).　While　AA　could　induce　the　redox　cycling　to　generate　electrocatalytic　oxidation　current　in　the　presence　of　ferrocene　methanol　(FcM).　When　capture　probes　hybridized　with　p53,　Nt.BstNBI　would　recognize　and　cleave　the　duplexes　and　p53　was　released　for　recycling.　Meanwhile,　the　biotin　group　dropt　from　the　electrode　surface　and　subsequently　SA-ALP　could　not　adhere　to　the　electrode.　The　signal　difference　before　and　after　cleavage　was　proportional　to　the　p53　gene　concentration.　Furthermore,　with　electrode　temperature　elevated,　the　Nt.BstNBI　and　ALP　activities　could　be　increased,　greatly　improving　the　sensitivity　and　efficiency　for　p53　detection.　A　detection　limit　of　9.5　×　10−17　M　could　be　obtained　(S/N　=　3)　with　an　electrode　temperature　of　40　°C,　ca.　four　magnitudes　lower　than　that　at　25　°C.　©　2023　Elsevier　B.V.