The feasibility of producing L-alanine via fermentation, using glycerol as the sole carbon source was investigated. The recombinant strain Escherichia coli B0016-050 lacking routes to metabolites like acetate, formate, ethanol, succinate, and lactate within its metabolic network served as the starting strain. The gene encoding L-alanine dehydrogenase, alaD, from Geobacillus stearothermophilus, which was controlled by the left promoter from phage λ (λ pL promoter), was used to replace the alanine racemase-encoding dadX gene in the genome of strain B0016-050 to generate the thermoregulatory L-alanine-producing strain B0016-060BC. Strain B0016-060BC carried out a two-phase fermentation (including a cell growth phase and an L-alanine-producing phase) with glycerol as the sole carbon source, and the results indicate that L-alanine production by strain B0016-060BC can be improved by initiating L-alanine production at the late exponential growth phase or by increasing the aeration rate during fermentation. After fermentation in a 5-L bioreactor, 63.64 g/L L-alanine was produced with an overall productivity and conversion rate of 1.91 g/L h and 62.89 g/100 g glycerol, respectively. Only small amounts of acetate (1.73 g/L) and other byproducts were synthesized. Therefore, efficient L-alanine production using glycerol as the sole carbon source was realized, providing an important reference for industrial application.