This dissertation focuses on the development of sheath-flow surface enhanced Raman (SERS) detection for liquid chromatography (LC), to enable online separation, detection and quantification of biomolecules at low concentrations in flow. The experiments reported here focused on the direct, label-free SERS aspects in analysis and identification of small biomolecules. The direct SERS detection is very straight forward and has been reported to be able to detect different classes of biomolecules ranging from nucleic acids, proteins, carbohydrates, and nucleotides to small molecule metabolites. The coupling of LC-SERS to a lab-built flow cell using hydrodynamic focusing is reported. The SERS cell is connected directly with the LC outlet for simultaneous post-separation detection. A mixture of model metabolites (thiamine, folic acid, and riboflavin) has been successfully separated, characterized and quantified using acetonitrile in the mobile phase provides an internal standard. The results demonstrated that sheath-flow SERS is provides improved detection of molecules compared to standard UV-Vis detectors. The LC-SERS system has been further explored to detect and quantify of three phosphorylated carbohydrate molecules: glucose 1-phosphate, glucose 6-phosphate and fructose 6-phosphate. An alkanethiol (hexanethiol) self-assembled monolayer is formed over the silver SERS-active substrate helps retain and concentrate the analytes of interest at the SERS substrate to improve the detection sensitivity significantly. Mixtures of 2 µM of phosphorylated carbohydrates in pure water as well as in cell culture media were successfully separated by HPLC, with identification using the sheath-flow SERS detector. The results suggested a tremendous potential for LC-SERS for targeted molecular identification in complex biological environment for metabolomics and other bioanalytical studies.The excellent sensitivity of sheath-flow SERS has provided a new detection technique for proteins study. For instant, the sheath-flow SERS has been successfully demonstrated to decipher the three different small ubiquitin like modifier (SUMO) proteins of a post-translational modifications process of proteins known as the SUMOylation. The differentiation of three SUMO proteins was achieved in a direct, label-free approach without the need of additional sample functionalization or affinity reagents. Additionally, we demonstrate that the identity of the SUMO tag on a protein can be determined.