Herein, we present a colorimetric detection method based on the surface-enhanced photochromic phenomenon of tungsten (VI) oxide (WO3) nanocolloid particles for α-amino acid (AA) molecules, including L-aspartic acid (Asp), L-glutamic acid (Glu), L-histidine (His), L-isoleucine (Ile), L-leucine (Leu), L-lysine (Lys), L-phenylalanine (Phe), and L-valine (Val). The UV-induced photochromic phenomena in the AA/WO3 binary aqueous systems were investigated using UV-Vis absorption spectrometry. The adsorption properties of the AA molecules on the surface of the WO3 nanocolloid particles have been identified using a combination of adsorption isotherm analysis and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. A good linear correlation between the concentration of the AAs adsorbed on the surface of the WO3 nanocolloid particles and the initial photochromic coloration rate in the corresponding UV-irradiated WO3 colloidal aqueous solution was obtained with over three orders of magnitude, indicating that the surface-enhanced photochromic phenomenon of the WO3 nanocolloid particle can be used to detect the AA molecules. In addition, based on the results of the UV-Vis absorption, ATR-FTIR, and adsorption isotherm analyses, we have experimentally demonstrated that the AA/WO3 binary aqueous system with inner-sphere adsorbed Ile, Leu, Lys, or Val molecules on the surface of the WO3 nanocolloid particles exhibits a more significant surface-enhanced photochromic phenomenon than the system with outer-sphere adsorbed Asp, Glu, His, or Phe molecules. The strong inner-sphere adsorption of the AA molecules successfully improved the limit of detection. This study provides valuable insights into a "label-free" colorimetric assay system based on the surface-enhanced photochromic phenomenon of the WO3 nanocolloid probe.