Microbioreactors are effective for high-throughput production of expensive products from small amounts of substrates. Lipases are versatile enzymes for chiral syntheses, and are highly activated when immobilized in alkyl-substituted silicates by the sol-gel method. For practical application of sol-gel immobilized lipases to a flow system, a microbioreactor loaded with a macroporous silica monolith is well suited, because it can be easily integrated with a chromatographic separator for optical resolution. We attempted to develop a microbioreactor containing a silica monolith-immobilized lipase. A nonshrinkable silica monolith was first formed from a 4:1 mixture of methyltrimethoxysilane (MTMS) and tetramethoxysilane (TMOS). It was then coated with silica precipitates entrapping lipase, derived from a 4:1 mixture of n-butyltrimethoxysilane (BTMS) and TMOS. As a result, monolith treated with the BTMS-based silicate entrapping lipase exhibited approximately ten times higher activity than nontreated monolith-immobilized lipase derived from the MTMS-based silicate, in transesterification between glycidol and vinyl n-butyrate in isooctane. A commercially available chiral column was connected in series to the monolith microbioreactor, and a pulse of substrate solution was supplied at the inlet of the reactor. Successful resolution of the racemic ester produced was achieved in the chromatographic column.