The use of membranes for on-line separations in flow-through dialyser as a part of a flow system is extremely useful for automated samples preparation. In this paper a method to couple the dialysis and the UV detection on-line of Vitamin B(12) from milk is proposed. Firstly, the milk samples were pre-treated with trichloroacetic and centrifuged (to eliminate proteins and fats) and later, using a dialyser coupled a flow-continuous manifold was possible dialyse the Vitamin B(12), which was monitored spectrophotometrically at 361nm. On the other hand, the milk samples were also dialysed on-line but without the acid treatment and the results were compared. The influence of various parameters, including the pump speed for both the donor and acceptor stream, dialysis time, donor and acceptor loop volume on dialysis efficiency was studied. The method was applied to different kinds of milk (skimmed and semi-skimmed milk, evaporated milk, lactose free milk and liquid and powder whole milk). The relative standard deviation (R.S.D.) of the proposed method was of 0.45% and the obtained dialysis percentage was of 5.8%. The proposed method very easy permit a pre-treatment of the sample, quick and on-line with the detection. The dialysis process permitted the pass of vitamin and avoided the pass of other analytes as proteins in the case of the milk samples without acid treatment.