Based on the oxidation-redox mechanism of naturally occurring NADH along with its metal coordination properties, biogenic AuPd nanoclusters are directly synthesized with low concentration of metal precursors, showing outstanding peroxidase mimicking activities. Ultrasmall bimetallic nanozymes can rapidly form under the reduction and the stabilization of NADH, and their physicochemical properties are closely associated to the molar ratio of [Na2PdCl4]/[HAuCl4]. Synergistic effects between Au and Pd play a particular role in both growth kinetics and catalytic performance of AuPd nanoclusters. The strong Au-Pd interactions remarkably adjust the electronic properties of both Au and Pd species in bimetallic NCs, thereby contributing to their superior enzymatic activities over two monometallic counterparts. Based on the peroxidase-like activity of AuPd nanoclusters at acidic pH, a colorimetric test is developed for monitoring acid phosphatase quantitatively, giving the limit of detection of 0.53 U/L. The linear response lies in the concentration range of 1–14 U/L. This method is also suitable for estimating ACP activity in biological fluid.