Although Blakeslea trispora has been used for industrial production of β-carotene, the effects of light and oxidative stress on its synthesis have not been fully clarified. The present study focuses on the effects of light and reactive oxygen species (ROS) on carotenoid synthesis and their multilevel regulation in B. trispora. Blue light significantly influenced the intracellular ROS levels, carotenoid contents, and transcription of carotenoid structural genes, while ROS levels were positively correlated with intracellular carotenoid contents and transcriptional levels of carotenoid structural genes. Blue light and ROS were both significant factors affecting carotenoid synthesis with a significant interaction between them. Irradiation by pulsed blue light and (or) addition of generating agents for active oxygen could partially compensate for the inhibition derived from the transcription inhibitor (dactinomycin) and translation inhibitor (cycloheximide) on the multilevel phenotype. Therefore, blue light and ROS act on the transcription and translation of carotenoid structural genes to promote the accumulation of carotenoid in B. trispora.