[Background] Bottleneck problems such as poor yield and low productivity had been hindering the progress in commercial production of polymalic acid from glucose by Aureobasidium pullulans. [Objective] To enhance polymalic acid yield and productivity from Aureobasidium pullulans BK-10 by optimizing the fermentation medium and conditions. [Methods] Single-factor experiment, Plackett-Burman design and orthogonal experiment were adopted to evaluate the importance of various fermentation medium ingredients and optimize kinds of fermentation conditions. A 5 L fermentor experiment was conducted to verify the above optimized conditions. [Results] The optimum fermentation medium consisted of 100 g/L glucose, 1.5 g/L urea, 0.20 g/L KH2PO4, 0.20 g/L ZnSO4, 0.05 g/L MgSO4, 0.75 g/L KCl, 30 g/L CaCO3, 0.01% Tween-80, the optimal fermentation temperature was found 26 °C and the optimal working volume was 50 mL of fermentation medium in a 250 mL flask. [Conclusion] Yield of polymalic acid from glucose reached 0.71 g/g with an increase of 18.33%, and the productivity was 0.89 g/(L·h) which was increased by 71.15% as compared with the unoptimized conditions. These results show great economical potential for commercial production of polymalic acid even or L-malic acid by fermentation from glucose.