Glycans and glycosylated biomolecules are directly involved in almost every biological process as well as the etiology of most major diseases. Hence, glycoscience knowledge is essential to efforts aimed at addressing fundamental challenges in understanding and improving human health. While much progress has been made, there remains an urgent need for new tools that can overexpress structurally uniform glycans and glycoconjugates in the quantities needed for characterization and that can be used to mechanistically dissect the enzymatic reactions and multi-enzyme assembly lines that promote their construction. To address this technology gap, we develop the cell-free synthetic glycobiology system as a simplified and highly modular framework to investigate, prototype, and engineer pathways for glycan biosynthesis and protein glycosylation outside the confines of living cells. First, we engineered a novel cell-free glycoprotein synthesis (CFGpS) system that seamlessly integrates protein biosynthesis with asparagine-linked (N-linked) or serine/threonine-linked (O-linked) protein glycosylation. This technology leveraged a glyco-optimized Escherichia coli strain to source crude extracts that were selectively enriched with glycosylation components. The resulting extracts enabled a one-pot reaction scheme for efficient and site-specific glycosylation of therapeutic proteins including biologically-active human erythropoietin. Next, we expanded the utility of the cell-free glycosylation system for an in vitro bioconjugate vaccine expression (iVAX) in lysates derived from detoxified, nonpathogenic E. coli. We demonstrated that iVAX synthesized vaccines against Franciscella tularensis subsp. tularensis (type A) strain Schu S4 conferred complete protection in an intranasal mouse model of F. tularensis infection. Finally, we developed a collection of the cell-free glycan remodeling modules, enabled by the creation of a glycosyltransferase high-level expression library. The glycan remodeling modules are facile and highly modular, providing an efficient platform for rapid biosynthesis of authentic, complex human N-glycans. Together, our cell-free synthetic glycosylation system effectively broadens the glycoengineering toolbox and is anticipated to facilitate fundamental understanding in glycoscience and make possible new applications in on-demand biomanufacturing of glycoprotein therapeutics and vaccines.