β-cryptoxanthin is the most commercially valuable carotenoid. This research aimed to investigate the effects of imidazole treatment on the growth and β-cryptoxanthin accumulation in Dunaliella tertiolecta. The results showed that imidazole reduced the biomass, chlorophylls and total carotenoids content of D. tertiolecta, but rapidly (4 days) and considerably increased the accumulation of β-cryptoxanthin. The combination treatment of imidazole with different light intensities could further promote the accumulation of β-cryptoxanthin. Under 8000lx light intensity, treatment with 500ppm imidazole for 4d is most conducive to the accumulation of β-cryptoxanthin in D. tertiolecta, in which condition the content of β-cryptoxanthin and single cell β-cryptoxanthin were 1.133 times and 1.273 times higher than that in 250 ppm imidazole group, and the expression levels of DtBHY and DtZEP genes was significantly increased. The findings in this research will provide valuable information for chemical inducers to enhance the accumulation of β-cryptoxanthin in microalgae and plants.