A series of homogeneous catalysts consisting of pyridinium propyl sulfobetaine (PPS), tungstophosphoric acid (TPA), and acetic acid (HOAc) have been synthesized and exploited for catalytic acetylation of glycerol (GL). Their acid properties were characterized by P-31 NMR of trimethylphosphine oxide (TMPO) as the probe molecule, and the effects of acidic strength, PPS/TPA, TPA/GL, and HOAc/GL ratios as well as reaction temperature on catalytic performances during acetylation reaction were investigated. These water-tolerable PPS-TPA-HOAc catalysts, which tend to segregate from glycerol acetate products to form distinct biphasic liquid layers spontaneously after the reaction, were found to be highly efficient and durable for acetylation reaction under continuous operation conditions. Typically, a complete GL conversion may be achieved with a superior glycerol triacetate (GTA) selectivity of 86-99%. Moreover, the unique self-separation biphasic characteristics of the catalyst system facilitate facile separation of products and recycling of catalyst, rendering practical industrial applications in acetylation of alcohol.