Vanda tricolor is an orchid species that has many advantages, some of which are due to its beautiful and various flowers of unique shape and size. Propagation of V. tricolor can be performed by in vitro culture.In vitro culture technique needs appropriate medium to support orchid growth. An algae species, Sargassum duplicatum, can be added into the medium, since it contains growth regulating substances, such as auxin and gibberellin, which are useful for seedling growth. This study aims to know the effect of S. duplicatum application into in vitro culture medium of V. tricolor on the seedling growth and to know the best application of the algaein increasing seedling growth. An experimental method was arranged in a Completely Randomized Design (CRD). The treatments were S. duplicatum weight of 0, 12, 24, 36, 48 and 60 g/L replicated three times respectively. The variable examined was seedling growth with parameters comprising day of new leaf emergence, leaf number, leaf length, day of new root emergence, root number, root length and plant height. Data were analyzed using ANOVA or F test with confidence level of 95% and 99%. LSD was carried out when F test showed significant difference. The results reveal that application of S. duplicatum extract has significant effect on the growth of V. tricolor seedling. The best application is of 36 g/L algae weight.