Glycoengineering aims to generate proteins with defined glycosylation. Single monosaccharide differences such as the absence of core fucosylation to the N-glycan in immunoglobulin G can cause a 100-fold increase in antibody-dependent cell cytotoxicity response. Production of glycoengineered proteins can be performed in vivo by genetic modification of host glycosylation enzyme expression pre-purification of the target protein, or in vitro using glycan modifying enzymes post-purification. While in vitro methods can be costly from using external enzymes and require lengthy incubation times, enzymatic reactions can be controlled to a degree unattainable in vivo. Here, we show the promise of an artificial Golgi column by performing in vitro glycosylation in an on-line column format with immobilised glycosyltransferase enzymes. By immobilising recombinantly expressed β-1,4-galactosyltransferase 1 via the poly-histidine purification tag, we utilised existing liquid chromatography systems to introduce protein, nucleotide sugar substrates and essential metal ion co-factor to the enzyme, mimicking the trans-Golgi network in the cell. The flow driven process promotes the encounter between the enzyme and substrates, enabling the enzymatic transfer of galactose onto unoccupied GlcNAc of acceptor glycans on the target protein substrate within minutes. With the enzymes immobilised in a column format, protein substrates can be continuously introduced to the enzymes which allows reusability of the column, creating a more cost-effective process compared to using free enzymes. Coupling to a liquid chromatography system enables accurate optimisation of the enzymatic reaction; we have shown that adjusting physical parameters such as column internal diameter, co-factor concentration, substrate concentration and flow rate make substantial changes to the efficiency of reaction completeness in this artificial Golgi column. Multiple enzymes can also be incorporated in sequence on-line. This form of glycoengineering shows promise for future industrial applications with the potential for large-scale glycan modification of valuable therapeutics and other glycoproteins.