Isochcrysis strains as versatile cell factories can produce varieties of high-valued bioproducts for food, nutraceuticals and pharmaceuticals. This work aimed to evaluate the ability of Isochrysis sp. to produce high-valued triacylglycerols (TAGs) for human milk fat substitutes (HMFs) and fucoxanthin by batch, fed-batch and semi-continuous cultivation modes in 50-L column photobioreactors. It was found that, Isochrysis cells by three cultivation modes had individual growth characteristics, nutrients consumption, and biochemical composition. In all cultivation modes, microalga cultivated by the semi-continuous cultivation at 10 % renewable rate achieves the highest TAGs and fucoxanthin productivities with 8.2 and 0.5 mg/(L·d), respectively. Additionally, the obtained results showed that the microalgal growth phase and the levels of nitrogen and phosphorus had distinct impacts on the deducting G value for microalgal TAGs produced by three cultivation modes. Based on the fatty acid composition and positional distribution, Isochrysis TAGs by the semi-continuous process at 10 % renewable rate had 73.0 deducting G value that were close to the ones by the batch cultivation (13th day) and fed-batch cultivation. Taken together, Isochrysis sp. was the promising marine microalga to concurrently produce sustainable high-valued TAGs for HMFs and fucoxanthin by semi-continuous cultivation in pilot-scale photobioreactors. This work provides a new marine microalgal platform to exploit Isochrysis-derived TAGs as human milk fat subsitutes for infant formulas and fucoxanthin for nutraceuticals and pharmaceuticals.