The gut microbiota, a complex community of microorganisms, plays a crucial role in gastrointestinal (GI) health, influencing digestion, metabolism, immune function, and the gut-brain axis. Dysbiosis, or an imbalance in microbiota composition, is associated with GI disorders, including irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), and colorectal cancer (CRC). Conventional microbiota analysis methods, such as next-generation sequencing (NGS) and nuclear magnetic resonance (NMR), provide valuable insights but are often expensive, time-consuming, and destructive. Raman spectroscopy (RS) is a non-invasive, cost-effective, and highly sensitive alternative. This analytical technique relies on inelastic light scattering to generate molecular "fingerprints", enabling real-time, marker-free analysis of microbiota composition and metabolic activity. This review explores the principles, sample preparation techniques, and advancements in RS, including surface-enhanced Raman spectroscopy (SERS), for microbiota research. RS facilitates identifying microbial species, analysing key metabolites like short-chain fatty acids (SCFA), and monitoring microbiota responses to dietary and therapeutic interventions. The comparative analysis highlights RS's advantages over conventional techniques, such as the minimal sample preparation, real-time capabilities, and non-destructive nature. The integration of RS with machine learning enhances its diagnostic potential, enabling biomarker discovery and personalised treatment strategies for GI disorders. Challenges, including weak Raman signals and spectral complexity, are discussed alongside emerging solutions. As RS technology advances, mainly through portable spectrometers and AI integration, its clinical application in microbiota diagnostics and personalised medicine is poised to transform GI healthcare, bridging microbiota research with practical therapeutic strategies.