The invention discloses a culture medium box set and application of the culture medium box set in rapid in vitro propagation of vitex rotundifolia. The culture medium box set comprises a culture medium for establishing an asepsis system, a culture medium for inducing and proliferating cluster buds, a culture medium for inducing adventitious buds, a culture medium for elongation inducing of the adventitious buds, a culture medium for rooting and a substrate formed by mixing yellow mud and peat soil with a volume ratio of 3 to 1. According to the culture medium box set disclosed by the invention, the adventitious bud generation path induced by flattening bud stem segments is discovered for the first time, and in vitro culture of the vitex rotundifolia is carried out by using the culture medium box set disclosed by the invention; the propagation rate of cluster buds can be up to 21.9, the average quantity of the adventitious buds per flattened explant can be up to 84.9, the elongation ofthe adventitious buds can be up to 3. 0cm, the rooting rate and average rooting number can respectively reach 70.5 percent and 86.4, and the survival rate of the adventitious buds can be up to 86 percent after being transplanted for 30 days. The culture medium box set has a good application prospect.