In this study, Raman spectroscopy was first used to study the linear relationship between Raman spectral intensities and citric acid concentrations in aqueous solution. By using the specific Raman band of 942 cm<sup>?1</sup>, concentrations of citric acid ranging from 2 to 20 mg/mL were observed linearly (<i>R</i><sup>2</sup> = 0.993), and the limit of detection was 1.0 mg/mL. Then, citric acid detection in clinical seminal plasma ultrafiltrate samples was performed, and the intensity of the Raman-specific peak demonstrates a good linear correlation (<i>R</i><sup>2</sup> = 0.946) with citric acid concentrations determined by the enzymatic method. Our results showed that Raman spectroscopy has the potential of being applied to detect concentrations of citric acid in seminal plasma in clinic.

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