The preparation of a novel biofuel denoted as Ecodiesel-100 from the partial 1,3-regiospecific alcoholysis of sunflower oil is reported. Pig pancreatic lipase (PPL) was employed in the reaction as both free and immobilised enzyme on sepiolite. The resulting biofuel is composed of fatty acid ethyl esters and monoglycerides (FAEE/MG) blended in a molar relation 2/1. The novel biofuel has similar physico-chemical properties compared to those of conventional biodiesel and/or petrodiesel, avoiding the production of glycerine as by-product.The biocatalyst was found to be strongly fixed to the inorganic support (87.5%). Nevertheless, the efficiency of the immobilised enzyme was reduced to less than half (42%) compared to that of the free PPL. Quantitative conversions of triglycerides and high yields to FAEE were obtained under mild reaction conditions (20–80 °C, oil/alcohol 2/1 v:v ratio and PPL 0.01–0.1% w/w of total substrate). The immobilised enzyme showed a remarkable stability as well as a great reusability (more than 11 successive reuses) without a significant loss of its initial catalytic activity. Both immobilised and free enzyme exhibited the same reaction mechanism, according to the coincidental results in the Arrhenius parameters (Ln A and Ea). The immobilised PPL was found to be very suitable for the continuous production of biofuel due to its facile recyclability from the reaction mixture.