The feasibility of L-alanine fermentation using glycerol as a sole carbon source was investigated. The recombinant strain Escherichia coli B0016-050 with deletions of acetate, formate, ethanol, succinate, and lactate synthesis routes served as the parent strain. The λ pL promoter and L-alanine dehydrogenase (derived from Geobacillus stearothermophilus) controlled by the λ pL promoter were used to replace the dadX gene (encoding L-alanine racemase) on the chromosome of strain B0016-050 to generate the thermoregulated L-alanine fermentation strain B0016-060BC. The fermentation by strain B0016-060BC was a two-phase process (consisting of a cell growth phase and an L-alanine synthesis phase) with glycerol as the sole carbon source, indicating that L-alanine production in strain B0016-060BC could be improved by initiating the production in the late exponential growth phase or by increasing air supplementation in the fermentation stage. Fermentation in a 5-L bioreactor yielded 63.64 g/L L-alanine, with overall productivity and yield of 1.91 g/L h and 62.89 g/100 g glycerol, respectively, and only a small amount of acetate (1.73 g/L) was produced as a byproduct. Therefore, the efficient production of L-alanine using glycerol as the sole carbon source is possible, providing an important reference for industrial applications.