Regulating morphology engineering and fermentation of Aspergillus oryzae makes it possible to increase the titer of L-malate. However, the existing L-malate-producing strain has limited L-malate production capacity and the fermentation process is insufficiently mature, which cannot meet the needs of industrial L-malate production. To further increase the L-malate production capacity of A. oryzae, we screened out a mutant strain (FMME-S-38) that produced 79.8 g/L L-malate in 250-mL shake flasks, using a newly developed screening system based on colony morphology on the plate. We further compared the extracellular nitrogen (N1) and intracellular nitrogen (N2) contents of the control and mutant strain (FMME-S-38) to determine the relationship between the curve of nitrogen content (N1 and N2) and the L-malate titer. This correlation was then used to optimize the conditions for developing a novel nitrogen supply strategy (initial tryptone concentration of 6.5 g/L and feeding with 3 g/L tryptone at 24 h). Fermentation in a 7.5-L fermentor under the optimized conditions further increased the titer and productivity of L-malate to 143.3 g/L and 1.19 g/L/h, respectively, corresponding to 164.9 g/L and 1.14 g/L/h in a 30-L fermentor. This nitrogen regulation-based strategy cannot only enhance industrial-scale L-malate production but also has generalizability and the potential to increase the production of similar metabolites. Key Points center dot Construction of a new screening system based on colony morphology on the plate. center dot A novel nitrogen regulation strategy used to regulate the production of L-malate. center dot A nitrogen supply strategy used to maximize the production of L-malate.