Abstract: Amino acid analysis has always been a crucial aspect of chemical and biological research due to its essential biological significance and diverse roles in living organisms, including catalyzing chemical reactions, signaling, and supporting cell structure. Mass spectrometry possesses significant advantages in terms of sensitivity and accuracy among various amino acid analysis methods. Liquid chromatography-mass spectrometry (LC-MS) is the most commonly used method for amino acid mass spectrometry analysis, requiring an electrospray ion source. While the electrospray ion source has many advantages, such as high sensitivity, high selectivity, simple operation, compatibility with various types of mass spectrometers, and a wide range of analyzable substances, its ionization efficiency for amino acids is limited, particularly for acidic amino acids. This limitation hinders further improvement of the detection sensitivity of analytical methods. A mass spectrometry method for determining 20 common amino acids was established based on a self-developed Polarity-Reversing Nano-Electrospray Ionization (PR-nESI) ion source. PR-nESI enhances detection by altering the high-voltage strategy during the nano-spray process. Compared to conventional Nano-Electrospray Ionization (nano-ESI) methods, PR-nESI significantly improves the detection signal-to-noise ratio, increasing up to tens of times. Moreover, PR-nESI exhibits strong desalting effects, effectively inhibiting the formation of metal ion adducts of amino acids, which significantly facilitates spectral identification, particularly for biomolecules in salt buffer systems. The amino acid detection method presented in this study holds promising future application prospects in clinical and food fields.