<p>Detection of small molecular metabolites in the body can reflect the overall health status of the human body, being essential for understanding metabolism and diagnosing diseases. For the detection and monitoring of small molecular metabolites in complicated biological systems, the sensitive detections of trace metabolites and their pathways have been pursued by different strategies. Mass spectrometry provides high sensitivity and specificity for metabolic studies, while commercial techniques normally require sample pretreatments to limit the multiple examining requirements. Fortunately, ambient mass spectrometry (AMS) can meet rapid clinical examinations due to its rapid and direct detection of biological molecules without or with fewer sample pretreatments. By virtue of AMS, the real-time and online monitoring of small molecular metabolites can be achieved for examining metabolism mechanisms and facilitating targeted interventions. This review summarizes the application of AMS in the monitoring of small molecular metabolites, including glucose, lipids, amino acids, nucleotides, and aldehydes, as well as examinations of reaction mechanisms in clinical applications. This would provide insights on constructing powerful diagnostic tools for clinical applications.</p>