Using a Cephalosporin C-producing strain Cephalosporium acremonium B-1822-S-04 as the starting strain, we screened four Cephalosporin C high-yield strains D-G-017, D-G-083, D-G-096, and D-G-133, with compound mutagenesis by UV and malonic acid. The titer of strain D-G-096 is as high as 6 816 mg·L∼(-1), which is 50.80% higher than that of the starting strain, and the high-yield characteristics is stable. The small-scale experiments in 50 L fermentor show that the titer of the strain D-G-096 reaches 29 270 mg·L∼(-1), which is 111.06% higher than that of the starting strain. It can be used as an excellent high-yield strain. The high-yield strains screened in this experiment and their application in small-scale experiment can provide high-quality strain resources and fermentation process guidance for later industrial production