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An effective and simple method for the fabrication of Ag nanosheets (NSs) was developed via citrate-assisted galvanic replacement at room temperature. The effects of reaction time, concentration of AgNO3 and citrate on the morphology and surface-enhanced Raman scattering (SERS) performance of Ag NSs were investigated systematically. The results indicated that citrate played a vital role on preparing uniform Ag NSs, because it could retard the reaction rate and control the direction of Ag growth. Furthermore, citrate was bound weakly toward Ag NSs and easily replaced by probe molecules, which opened a new avenue for the fabrication of clean SERS substrates. In addition, the Ag NSs substrate exhibited a high sensitivity in SERS detection and the analytical enhancement factor was up to 7.93 × 105 with the minimum detected concentration of R6G as low as 10−8 M. Moreover, the color mapping was relatively uniform and the relative standard deviation was below 15%, revealing that the SERS performance of Ag NSs substrate was highly reproducible. Consequently, we believe this citrate-directed galvanic replacement could regulate the morphology and produce clean and highly active SERS substrates, which would be potentially useful for its practical application. [ABSTRACT FROM AUTHOR]