The 14 alpha-hydroxysteroids have specific anti-gonadotropic and carcinolytic biological activities and can be produced by microbial biotransformation. The steroid 11 beta-/14 alpha-hydroxylase P-450(lun) from Cochliobolus lunatus is the only fungal cytochrome P450 enzyme identified to date with steroid C14 hydroxylation ability. Previous work has mainly revealed the 11 beta-hydroxylation activity of the P-450(lun) towards cortexolone (RSS) substrate; however, the potential steroid 14 alpha-hydroxylation activity of this enzyme, especially for androstenedione (AD) substrate, has not yet conducted in-depth testing. In this work, we further tested the steroid 14 alpha-hydroxylation activity of the P-450(lun) towards RSS and AD in the Saccharomyces cerevisiae system. We demonstrated that P-450(lun) functions as the specific 14 alpha-hydroxylase towards the AD substrate (regiospecificity > 99%); however, it showed a poor C14-hydroxylation regiospecificity (around 40%) for the RSS substrate. In addition, through transcriptome analysis combined with gene functional characterizations, we also identified and cloned the gene for the P-450(lun)-associated redox partner CPRlun. Finally, through codon optimization, knockout of genes for the side reactions related enzymes GCY1 and YPR1, and increasing copies of the P-450(lun) and CPRlun, we developed a recombinant S. cerevisiae biocatalyst based on the C. lunatus steroid 14 alpha-hydroxylation system to produce 14 alpha-hydroxysteroids. Initial production of 14 alpha-OH-AD (150 mg/L day productivity, 99% regioisomeric purity, and 60% w/w yield) and 14 alpha-OH-RSS (64 mg/L day productivity, 40% regioisomeric purity, and 26% w/w yield) were separately achieved in shake flasks; these results represent the highest level of 14 alpha-hydroxysteroid production in the current yeast system.