Levulinic acid is a feasible platform chemical derived from acid-catalyzed hydrolysis of lignocellulose. The conversion of this substrate to (S)-gamma-valerolactone ((S)-GVL) was investigated in a chemo-enzymatic reaction sequence that benefits from mild reaction conditions and excellent enantiomeric excess of the desired (S)-GVL. For that purpose, levulinic acid was chemically esterified over the ion exchange resin Amberlyst 15 to yield ethyl levulinate (LaOEt). The keto ester was successfully reduced by (S)-specific carbonyl reductase from Candida parapsilosis (CPCR2) in a substrate-coupled cofactor regeneration system utilizing isopropanol as cosubstrate. In classical batch experiments, a maximum conversion of 95 % was achieved using a 20-fold excess of isopropanol. Continuous reduction of LaOEt was carried out for 24 h, and a productivity of more than 5 mg (S)-ethyl-4-hydroxypentanoate (4HPOEt) per mu g CPCR2 was achieved. Afterwards (S)-4HPOEt (> 99%ee) was substituted to lipase-catalyzed lactonization using immobilized lipase B from Candida antarctica to yield (S)-GVL in 90 % overall yield and > 99%ee.