Lung cancer is one of the most common malignancies and the leading cause of cancer-related death worldwide. Among the technologies suitable for the rapid and accurate molecular diagnosis of lung cancer, ambient mass spectrometry (AMS) has gained increasing interest as it allows the direct profiling of molecular information from various biological samples (e.g., tissue, serum, urine and sputum) in real-time and with minimal or no sample pretreatment. This minireview summarizes the applications of AMS in lung cancer studies (including tissue molecular identification, the discovery of potential biomarkers, and surgical margin assessment), and discusses the challenges and perspectives of AMS in the clinical precision molecular diagnosis of lung cancer.