4-Octyl itaconate is a novel antiviral and immunoregulatory small molecule showing great potential in the treatment of various autoimmune diseases and viral infections. It is difficult to selectively esterify the C4 carboxyl group of itaconate acid via one-step direct esterification using chemical catalysts, while the two-step route with itaconic anhydride as an intermediate is environmentally unfriendly and costly. This research investigated the one-step and green synthesis of 4-octyl itaconate through the structure control of lipase, obtaining 4-octyl itaconate with over 98% yield and over 99% selectivity. Multiscale molecular dynamics simulations were applied to investigate the reaction mechanism. The cavity pocket of lipases resulted in a 4-octyl itaconate selectivity by affecting distribution of substrates toward the catalytic site. Toluene could enhance monoesterification in the C4 carboxyl group and contribute to a nearly 100% conversion from itaconate acid into 4-octyl itaconate by adjusting the catalytic microenvironment around the lipase, producing a shrinkage effect on the channel.