The fabrication and on-chip integration of surface-enhanced Raman scattering (SERS) optrodes are presented. In the optrode configuration, both the laser excitation and the back-scattered Raman signal are transmitted through the same optical fiber. The SERS-active component of the optrode was fabricated through the self-assembly of silver nanoparticles on the tip of optical fibers. The application of SERS optrodes to detect dyes in aqueous solution indicated a limit of quantification below 1 nM, using nile blue A as a molecular probe. Using the optrode-integrated microfluidic chip, it was possible to detect several different dyes from solutions sequentially injected into the same channel. This approach for sequential detection of different analytes is applicable to monitoring on-chip chemical processes. The narrow bandwidth of the vibrational information generated by SERS allowed solutions of different compositions of two chemically similar dyes to be distinguished using a dilution microfluidic chip. These results demonstrate the advantages of the SERS-optrode for microfluidics applications by illustrating the potential of this vibrational method to quantify components in a mixture.