The production of many secondary metabolites such as carotenoids is influenced by the type of carbon and nitrogen sources and C:N ratio applied in culture medium. The present study discusses the role of C:N ratio and screening of carbon and nitrogen sources using mixture analysis design in carotenoids production by Blakeslea trispora. The C:N ratios of 20, 40, and 60 with six nitrogen sources were evaluated. Results indicated that limitation of nitrogen source (C:N of 60) could improve carotenoids production. Six nitrogen and carbon sources were then screened using mixture analysis design. The most effective nitrogen and carbon sources were soybean powder and glucose, respectively. The productivity of carotenoids (983.8 ± 31.5 mg/L) based on consumed nitrogen and carbon source was 189.10 mg/g soybean powder and 19.66 mg/g glucose. Mixture analysis design indicated single carbon and nitrogen source were more effective than a mixture of sources for carotenoids production.