The invention relates to a fermentation process for preparing cephalosporin C and a fermentation medium used in the fermentation process. The fermentation process for preparing the cephalosporin C comprises the following steps: firstly, taking cephalosporium acremonium, activating, inoculating the activated cephalosporium acremonium to a seed culture medium, and culturing for 60-80 hours at the pH value of 6.8-7.2 at the temperature of 27-30 DEG C for preparing seeds; secondly, inoculating the seeds obtained in the step one to the fermentation medium, and fermenting for 110-150 hours at the pH value of 5.2-5.8 at the temperature of 24-29 DEG C; thirdly, carrying out centrifugal separation on fermentation liquor obtained in the step two, and taking supernatant liquid at the upper layer, so as to obtain cephalosporin C supernatant liquor. The fermentation process for preparing the cephalosporin C has the advantages that aminoadipic acid is added into the fermentation medium, a nutrient composition formula of the fermentation medium and fermentation technological conditions are optimized, biosynthesis capacity of the cephalosporin C is enhanced, fermentation titer is obviously increased, titer of the cephalosporin C is 38061-44837 microgram/mL, and quality of the cephalosporin C is not influenced, so that the fermentation process for preparing the cephalosporin C has a broad industrial application prospect.