Sensitive detection strategies for enzymes and their inhibitors not only have critical significance in clinical diagnosis but can be extended to food and environment analysis. Here, bimetallic AgPd alloy nanoflowers were synthesized using carbon dots as the reductant via a two-step reduction method. The AgPd bimetallic nanoflowers exhibit ascorbate oxidase mimetic activity, which can rapidly catalyze the oxidization of ascorbic acid to dehydroascorbic acid (DHA). The DHA can then react with o-phenylenediamine (OPD) to produce 3-(1, 2-dihydroxyethyl)furo[3,4-b] quinoxalin-1(3H)-one (DFQ), accompanying with the characteristic fluorescence peak at 440 nm for DFQ. Alkaline phosphatase catalyzes l-ascorbyl-2-phosphate to generate AA, while organic phosphorus pesticides (OPPs) such as chlorpyrifos can inhibit the activity of alkaline phosphatase. By utilizing the enzyme-nanozyme tandem catalytic reaction, the fluorescence of the system varies in the presence of different concentrations of ALP or OPPs, which can be used to detect ALP in serum and OPPs in vegetables and fruits.