Multiplexing of molecules with high sensitivity and selectivity is an essential technology in various fields. Detection of multiple species can significantly reduce analytical time and help provide thorough information from a single sample in applications such as diagnostics, pollution tests, and food safety. So far, various sensor technologies based on electrochemical response and optical signals have been developed to achieve multiplex detection. However, single electrochemical sensing components lack selectivity and thus fabrication of sensor arrays or data processing such as principle component analysis (PCA) is an inevitable step. Recently, reporter-based surface-enhanced Raman spectroscopy (SERS) has drawn much attention as a sensing technology for multiplex detection of trace-amount molecules. Despite its single-molecule level sensitivity and characteristic spectra of Raman reporter molecules, reporter-based SERS still faces the challenge of false positive response. In this thesis, multiplex detection via direct signal of target molecules is achieved by functionalizing aptamers onto SERS substrates with highly dense hot spots. Single-stranded DNA aptamers were anchored onto 3-dimensional plasmonic nanostructures consisted of well-ordered 20 nm Au nanorods by covalent thiolate bonds. The resulting aptamer-functionalized SERS substrates were able to selectively multiplex 3 different small molecules down to the nM level and also perform quantitative analysis for mixed samples of various compositions with high reproducibility.