Cross-talk phenomenon in dual-labeled fluorescent microarray scanning is analyzed from cross-excitation and cross-emission. It is turned out that the spectral overlap of the fluorophores is crucial for cross-talk error, and this error can be corrected by an image subtraction method. The experiment was successfully applied to separate the Cy3 channel and the Cy5 channel in microarray scanning. The cross-talk error was reduced from more than 1% to about 0.1%.