Objective To study the influencing factors of digital image white balance of the biological microscope and the way to obtain optimized image.Methods Color temperature meter was used to measure the influences of the factors and their correlations, and SASS software was employed for statistical analysis.Results Object lens might increase the color temperature by 200 to 300 K, LBD color filter could enhance it by 400 to 1 800 K. Reducing field stop by 90% might decrease color temperature by 400 K. ND6 filter, ND25 filter and aperture diaphragm had no influences on color temperature.Conclusion The electric and optical parameters of biological microscope have to be adjusted to gain proper white balance for digital image.