A new method, using sudan I as internal standard to determine the content of lycopene or beta-carotene in samples, was developed. According to UV-vis absorption spectra, sudan I, lycopene and beta-carotene all had absorption peaks at 450 nm. They could be separated absolutely by high-performance liquid chromatography (HPLC) with retention time of 2.7, 6.6 and 10.1 min, respectively. The related equations between sudan I and lycopene or beta-carotene content were obtained and verified by determining the content of lycopene or beta-carotene in Blakeslea trispora cells. The relative error was below 1.4% for determining lycopene content and below 1.9% for beta-carotene. Intra-day variability for lycopene determination was less than 3.4% and for beta-carotene was less than 1.4%. The mean recovery of lycopene or beta-carotene was 96.1 and 97.9%, respectively.