Lignocellulosic biomass can be valorized by fast pyrolysis, generating a bio-oil with levoglucosan (BoE), anhydrous sugar applied as building block for several compounds of great industrial interest. In this work we demonstrated for the first time the direct transesterification of BoE with different acyl donors by free and immobilized lipases. Influence of acetonitrile (ACN) and cyrene (CYE) in the stability of lipases from Candida rugosa and Candida antarctica B by differential scanning Nano fluorimetry (NanoDSF), at substrate concentrations of 20–300 mM as well as the alkyl chain effect for synthesis of the carbohydrate fatty acid esters (CFAEs) were carried out at 55 °C. Best results were obtained with Novozym® 435 in ACN and ethyl octanoate (C8) with 74% of conversion against 58% in CYE and ethyl palmitate (C16). In general it was observed that increasing on hydrophobicity of acyl donor contributed to greater regioselectivity for the hydroxyl bound to carbon 4 with up to 87% for C16 in ACN. Thermostability profile of CalB and CR suggested higher stabilty in CYE with values of Tm = 61 °C for CalB and Tm = 53 °C for CR. Finally, the kinetic parameters were obtained through the Michaelis-Menten constants generated by the double-reciprocal plot and indicated an inhibition by the LG substrate. Moreover, catalytic efficiency data suggest higher enzyme-substrate affinity in CYE.