The invention provides an amplification culture method for producing ethanol microorganisms by virtue of co-fermentation of C5 and C6. The method comprises the following steps: inoculating a seed solution obtained by culturing the microorganisms to an amplification culture medium; performing amplification culture on the microorganisms by virtue of at least one stage of an amplification culture step; and adjusting the oxygen supply concentration in the process of amplification culture to realize the amplification culture of the microorganisms.