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Small-sized monodisperse Pd-Ag alloy nanocrystals (NCs) are synthesized via a solid-liquid and solid-solid phase chemical route, i.e., sequential reduction of Pd(NO3)2 and AgNO3 solid precursors in the liquid mixture of dodecylamine and 1-octadecene, followed by fusion of formed Pd and Ag NCs at 250 °C. By controlling the addition sequence and molar ratio of the metallic precursors, a series of Pd-Ag alloy NCs, including Pd5Ag, Pd2Ag, PdAg, PdAg2, and PdAg5, is obtained. The alloy NCs are highly crystallized and exhibit a strong atomic ensemble in addition to component-dependent electronic effects. Pd2Ag NCs have unique structure and electronic properties, showing a much faster electron transfer process at a modified glassy carbon electrode interface compared with that of other alloys. Therefore, the Pd2Ag NCs are chosen as the electrocatalyst to evaluate the performance of Pd-Ag nanoalloy and a novel non-enzymatic glucose biosensor is fabricated. The biosensor exhibits an acceptable reproducibility, a good stability and low interferences, which can be used to examine glucose in clinic blood serum samples. This work provides a simple multiphasic reaction system to synthesize binary alloy NCs with well-controlled componential ratio and opens the avenue to utilize them in biosensing or other advanced technological fields. [ABSTRACT FROM AUTHOR]