The production of β-carotene from a synthetic medium by Blakeslea trispora in fed-batch culture was investigated. A maximum β-carotene concentration of 85.0 mg L−1 with productivity of 0.16 mg L−1 h−1 and specific β-carotene production rate of 0.01 mg g−1 h−1 was obtained by feeding the cells constantly with olive oil, cottonseed oil, soybean oil, antioxidant, and low concentration of growth factors at feeding rate of 4.2 mL h−1 from the beginning of the fermentation. In this case, the fed-batch culture supported high values of biomass dry weight (11.0 g L−1) and sugar utilization (0.976 g g−1). The morphology of the fungus was studied during growth in fed-batch fermentation system using an image analysis system. Zygospores are the morphological forms, which are responsible for the production of the pigment. The highest percentage of zygospores (11.44%) was correlated with the highest percentage of intracellular β-carotene (0.72%) in the total biomass dry weight. Moreover, high percentages of vacuolated hyphae, evacuated cells, and degenerated hyphae of the microorganism were observed. This was due to the formation of high amount of H2O2 by exposure of the microorganism to high dissolved oxygen concentration.