A feeding experiment with 40 lactating Holstein cows and 4 dietary treatments was conducted to investigate supplementation with different levels of alcohol fermented feed to the TMR on lactating performance, blood metabolites, milk fatty acid profile and cholesterol concentration of blood and milk. Forty Holstein lactating cows ($106{pm}24$ d post-partum; mean${pm}$SD) were distributed into four groups and randomly assigned to one of four treatments with each containing 10 cows per treatment. The treatment supplemented with TMR (DM basis) as the control (CON), and CON mixed with alcohol-fermented feeds (AFF) at a level of 5%, 10% and 15% of the TMR as T1, T2 and T3, respectively. Dry matter intake and milk yield were not affected by supplementation of AFF. An increased 4% FCM in the milk occurred in cows fed T3 diet compared with CON, while T1 and T2 diets decreased 4% FCM in a dose dependent manner. Supplementation of AFF increased the concentration of albumin, total protein (TP), ammonia, and high density lipoprotein-cholesterol in serum compared with CON. In contrast, supplementation with AFF clearly decreased concentration of blood urea nitrogen (BUN) and total cholesterol (TC) compare with CON. AFF supplementation increased the proportion of C18:1n9 and C18:2n6 compared to CON. A decrease in the concentration of saturated fatty acid (SFA) for T1, T2 and T3 resulted in an increased unsaturated fatty acid (USFA) to SFA ratio compared to CON. Concentration of cholesterol in milk fat was reduced in proportion to the supplemental level of AFF. Feeding a diet supplemented with a moderate level AFF to lactating cows could be a way to alter the feed efficiency and fatty acid profile of milk by increasing potentially human consumer healthy fatty acid without detrimental effects on feed intake and milk production. A substantially decreased cholesterol proportion in milk induced by supplementation AFF suggests that alcohol fermented feed may improve milk cholesterol levels without any negative effects in lactating cows.