Biotin and vitamin B₁₂ (VB₁₂) play an important role in regulating activity of hepatic gluconeogenesis related enzymes for the periparturient cows and hence had positive effects on health status and productivity of lactating dairy cows. However, their interactive effects remain unclear. Thus, this study was conducted to investigate the interactive effects of introducing biotin and VB₁₂ on health status and productivity in dairy cows. Forty-eight multiparous pre-partum dairy cows were allocated into 12 blocks based on parity (2.75 ± 0.89, mean ± SD) and daily milk yield (31.2 ± 3.28, mean ± SD kg/d), and were then randomly assigned to one of the 4 treatments: basal diet without, and with 30 mg/d dietary biotin, intramuscular injection of VB₁₂ at 10 mg/week once, or biotin addition (30 mg/d) plus VB₁₂ injection (10 mg/week). Treatments started from 3-week pre-partum (expected) to 8-week post-calving. Dry matter intake and milk productivity (yield and composition) were recorded weekly after calving. Blood from coccygeal vein were sampled biweekly after calving to determine serum nonesterified fatty acids, β-hydroxybutyric acid, and superoxide dismutase. Data were analyzed using the MIXED procedure of SAS. Body weight were measured at the beginning and end of the study. Injection of VB₁₂, but not dietary biotin, significantly increased feed intake and yield of milk, milk protein and lactose (P < 0.01), compared with the cows fed the basal diet. However, no interactive effects (P > 0.05) were observed on dry matter intake and milk yield between biotin and B₁₂. Serum nonesterified fatty acids, β-hydroxybutyric acid, and superoxide dismutase and body weight change did not differ across all the treatments (P > 0.05). In conclusion, biotin and VB₁₂ had limited interactive effect on dairy productivity. Introducing VB₁₂ in pre-partum dairy cows can increase milk yield rapidly without body weight loss, and is potentially beneficial to whole lactation cycle.