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For accurate experimental and theoretical research, the preparation of nanocrystrals with regular morphology is of significant importance. In this work, we investigated a facile and effective route for generating highly spherical Au@Ag nanospheres (NSs) with tuneable size and uniform morphology at room temperature. The aqueous synthesis mainly involved seed-mediated growth method together with oxidation etching employing sodium hypochlorite (NaClO) as etching agent. The termination of the etching reaction with NaClO as etching agent was simply related to the amount of NaClO and had no connection with time. Thus Au@Ag NSs with controllable diameters in range from 24 to 87 nm were prepared only by varying the amount of NaClO added into the solutions of Au@Ag nanocubes. Additionally, combined with interface self-assembly technique, Au@Ag NSs were assembled into densely arranged two-dimensional (2D) monolayer film. Moreover, the SERS performance of these monolayers were evaluated by calculating the analytical enhancement factor using crystal violet as probe molecule. The AEF increased obviously as the diameter of Au@Ag NSs went up, and the maximum AEF could reach to 0.94 × 107 at the laser excitation wavelength of 785 nm. Besides, the electromagnetic field distribution for Au@Ag NSs array were also confirmed by Mie theory and FDTD solutions software and the results revealed the similar trend with the experimental results. In general, this 2D assemblies in term of high quality Au@Ag NSs have broad prospects to act as promising candidates for SERS analytical sensor and other applications. [ABSTRACT FROM AUTHOR]