The aim of this study was to investigate the influence of conversion options in Photoshop on pre-press colour separation. Firstly, the basic principle of the conversion option in Photoshop was explained, and different conversion option settings were combined to conduct the pre-press color separation test, and then the test results were evaluated subjectively and objectively. Without conversion setting of black point compensation, tone merged seriously in shade area. Among the different colorimetry rendering intents, relative colorimetric rendering intent conversion had the smallest average value and standard deviation of color difference, while perceptual rendering intent had the widest color gamut range. Each conversion option had its own adaptive mapping purpose for the level or color conversion of the pre-press file. Users need to understand the basic principle and function before setting the conversion options according to the original pattern or the user preference.