The present study aimed to evaluate the effect of Laminaria japonica extract (LJE) supplementation on lactation performance, plasma biochemistry, immunity, antioxidant status, rumen fermentation parameters and rumen microbiota of dairy cows. The experiment was conducted in a one-way completely randomized design. 32 healthy lactating Holstein dairy cows with similar body weight, milk yield, parity and days in milk were selected and randomly assigned to 2 groups of 16 cows each. The cows in the control group (CON group) fed with a basal diet, and the other group (LJE group) received the CON diet supplemented with LJE at 30 g per cow per day. The entire trial includes a 12-day adaptation and a 30-day trial period. Compared with the CON group, the diet supplemented with LJE increased dry matter intake (DMI), milk yield, and milk protein content of dairy cows, whereas it had no significant effects on milk fat, lactose, somatic cell counts, and total solids, and it had no significant effects on the plasma biochemical indices. Moreover, the LJE group had significantly higher levels of immunoglobulin A (IgA), immunoglobulin G (IgG), and immunoglobulin M (IgM) in plasma, and had lower malondialdehyde content than those of the CON group. The total volatile fatty acid level increased in the rumen of dairy cows. The LJE diet increased the relative abundance of the phylum Firmicutes and the genera Succiniclasticum, Sphaerochaeta, Mucispirillum, and Akkermansia. In conclusion, adding LJE in feed can enhance the immunity of dairy cows, and reduce the level of lipid peroxidation in dairy cows, and improve the DMI, milk yield, and milk quality, which is of great significance to realize the healthy breeding of dairy cows.