Nanozymes　are　a　series　of　elaborately　designed　nanomaterials　that　can　mimic　the　catalytic　sites　of　natural　enzymes　for　reactions.　Bypassing　the　tedious　design　and　preparation　of　nanomaterial,　in　this　work,　we　report　on　a　novel　just-in-time　production　system　of　copper　hexacyanoferrate　nanoparticles　(CHNPs),　which　act　as　an　oxidase-mimicking　nanozyme.　This　system　can　rapidly　produce　CHNPs　nanozyme　on　demand　by　simply　mixing　Cu(II)　with　potassium　hexacyanoferrate(III)　(K3[Fe(CN)6]).　It　is　found　that　once　K3[Fe(CN)6]　is　reduced　to　K4[Fe(CN)6],　the　formation　of　CHNPs　is　inhibited.　Therefore,　the　just-in-time　production　system　of　CHNPs　was　coupled　with　alkaline　phosphatase　(ALP)　to　construct　an　enzyme-controllable　just-in-time　production　(ECJP)　system,　in　which　ALP　could　inhibit　the　production　of　by　catalyzing　the　hydrolysis　of　ascorbic　acid　2-phosphate　(AAP)　to　generating　ascorbic　acid　(AA).　The　ECJP　system　is　then　used　to　probe　the　activity　of　ALP　by　employing　2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonate)　(ABTS)　as　the　chromogenic　substrate,　and　a　detection　limit　of　0.003　U　L−1　was　achieved.　Moreover,　by　adapting　ALP　as　the　enzyme　label,　an　ECJP　system-based　colorimetric　immunoassay　protocol　was　established　for　sensitive　detection　of　aflatoxin　B1　(AFB1),　and　a　detection　limit　as　low　as　0.73　pg　mL−1　was　achieved.　The　developed　immunoassay　method　is　successfully　applied　to　the　detection　of　AFB1　in　peanut　samples.　The　operation　of　ECJP　system　is　quite　simple　and　the　coupling　of　ALP　with　CHNPs　nanozyme　can　arouse　dual　enzyme-like　cascade　signal　amplification.　So,　we　believe　this　work　can　offer　a　new　perspective　for　the　development　of　nanozymes-based　biodetection　methods　and　colorimetric　immunoassay　strategies.　©　2022　Elsevier　B.V.