The effect of moderate pulsed electric fields (MPEF) strength on autolysis of Saccharomyces cerevisiae was evaluated in this study. After exposure to MPEF with intensity of 7 kV cm−1 for 4 ms, the integrity of the cell wall was obviously destroyed and the inactivation of S. cerevisiae reached 99.43%. During the subsequent 42‐h autolysis process, the release of free α‐amino nitrogen of MPEF‐treated cells, as well as extracellular protease activity, was significantly (P < 0.05) higher than that of untreated cells. Moreover, exposure to 7 kV cm−1 led to an increase of the total amino acid of 149.36%. In particular, the content of aspartic acid and glutamic acid which are important umami flavour precursors increased by 232.55% and 209.40%, respectively. These results indicate that MPEF will be an effective method to accelerate autolysis of S. cerevisiae for obtaining high‐quality yeast extract as flavour enhancers and nutrition supplements.