Andrographolide, a diterpene lactone from Andrographis paniculata, has multiple pharmacological activities. And its acetylated derivatives showed improved antitumour activities. Herein, a total of ten lipases and four proteases were tested for regioselective acylation(s) of andrographolide by vinyl acetate in acetone. Although the enzyme activity was markedly source-dependent, the highest conversion with better initial reaction rate was achieved with immobilized Candida antarcticalipase B (Novozym 435), which displayed higher operational stability. The regioselective acylation at the 14-hydroxyl of the lactone was established by a combination of spectroscopic methods including ¹H and ¹³C NMR, DEPT, ESI-MS and FTIR spectra of the transformed product. The present work accommodated a feasible enzymatic approach for preparing andrographolide monoester, which possesses obvious advantages over the multi-step chemical process in terms of excellent regioselectivity, simplicity, environmental friendliness and mild reaction conditions.