We reported the significant effect of auxin and gibberellin on mixoploid cultures created by colchicine in Dunaliella salina. Polyploidy induction increased growth and the amount of all biochemical parameters measured in this work including chlorophyll, carotenoid, starch, glycerol, sugar, and protein. Treatment with colchicine 0.1%, which resulted in 58.26% of polyploid cells, had a better effect on increasing the amount of analyzed parameters. Auxin increased the amount of all measured parameters except protein. Low concentrations of auxin (1 and 10 µM) caused an increase in growth and the amount of chlorophyll, carotenoid, sugar, starch, glycerol, and protein in the cells treated with colchicine. Gibberellin significantly increased the amount of the mentioned parameters in a concentration‐dependent manner. In cultures treated with colchicine, additive effects of gibberellin were observed in glycerol, protein, starch, and sugar content. Our results showed that the use of phytohormones such as auxin and gibberellin can be a good way to increase the biochemical value of algal polyploid cell biomass.