Genetic engineering of microalgae to accumulate high levels of medium-chain-length fatty acids (MCFAs) represents an attractive strategy to improve the quality of microalgae-based biodiesel， but it has thus far been least successful. We demonstrate that one limitation is the availability of fatty acyl-acyl carrier protein (ACP) substrate pool for acyl-ACP thioesterase (TE). A combinational expression platform that involved plant lauric acid-biased TE (C12TE) and MCFA-specific ketoacyl-ACP synthase (KASIV) increased lauric acid (C12:0) and myristic acid (C14:0) accumulation by almost sevenfold and fourfold， respectively， compared with native strain. These findings suggest a platform for further investigation into the enlargement of MCFA acyl-ACP substrate pool as an approach to sustainably improve quality of microalgae-based biodiesel with regard to MCFA production.