Yarn-dyed fabric (YDF) is increasingly utilized in the textile industry owing to its excellent color fastness and reduced environmental impact from dyeing effluent. However, YDF's enzyme desizing is hindered by leached dyes inhibiting α-amylase. As inhibition is dye-dependent, elucidating the competitive mechanism is crucial for optimizing efficiency and minimizing enzyme consumption. This study investigated the effect of direct yellow 50 (DY-50), reactive yellow-K (RY-K), methylene blue (MB) and indigo on α-amylase activity at varying concentrations. α-Amylase samples exposed to dyes were analyzed using Fourier transform infrared spectroscopy, ultraviolet-visible spectrophotometry (UV-Vis), intrinsic fluorescence spectroscopy, sulfhydryl group content determination, zeta potential and circular dichroism spectroscopy to elucidate structural alterations and the underlying inhibition mechanisms. The results indicated that all four dyes negatively impacted α-amylase activity via mixed competitive inhibition. DY-50 exhibited the most potent inhibitory effect on α-amylase, inducing a 25 % activity loss after 60 min of exposure. Comparatively, activity losses were milder with indigo (20.7 %), RY-K (15.9 %), and MB (10.6 %). Beyond inhibiting starch-enzyme contact, dye exposure induced structural changes in α-amylase, including altered hydrogen bonding, exposure of hydrophobic groups, decreased α-helix content (from 19.1 % to 15.9 %), and increased β-sheet content (from 29.5 % to 33.2 %), collectively contributing to the activity loss of α-amylase. Elucidating these inhibition mechanisms offers vital strategies to optimize α-amylase utilization in YDF desizing, thereby boosting enzymatic efficiency and industrial economics.