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Single-drop microextraction (SDME) was coupled with surface-enhanced Raman scattering (SERS) to provide sample extraction and pre-concentration for detection of analyte at low concentrations. A gold nanohole array substrate (AuNHAS), fabricated by interference lithography, was used as SERS substrate and para-mercaptobenzoic acid (p-MBA) was tested as a probe molecule, in the concentration range 10−8−10−4 mol L−1. With this approach, a limit of 10−7 mol L−1 was clearly detected. To improve the detection to lower p-MBA concentration, as 10−8 mol L−1, the SDME technique was applied. The p-MBA Raman signature was detected in two performed extractions and its new concentration was determined to be ~4.6 × 10−5 mol L−1. This work showed that coupling SDME with SERS allowed a rapid (5 min) and efficient pre-concentration (from 10−8 mol L−1 to 10−5 mol L−1), detection, and quantification of the analyte of interest, proving to be an interesting analytical tool for SERS applications.