During glycerol fed-batch fermentation, the process could be divided into multiple equal subintervals, and the feed operation was performed in pulse form at the start of each subinterval. Based on the macrokinetic models, the multipulse feed strategy for both glucose and corn steep slurry was determined by a general nonlinear optimization approach to maximize the final glycerol productivity and still control the residual glucose at a low concentration. The experimental results in a 600-mL Airlift Loop Reactor showed that the tested data with this strategy agreed well with the corresponding model prediction, and that the feed mode with nonlinear optimization could improve the glycerol productivity significantly compared with those determined just by limited experimental optimization in previous studies.