To maximize the final production yield and control the residual glucose at a low concentration during glycerol production by fermentation, optimal and sub-optimal temperature control strategies were investigated. The optimal control strategy of the culture temperature was determined precisely by Pontryagin's Maximum Principle. By dividing the fermentation into multiple equal subintervals and optimizing the constant temperatures of all subintervals by a general static constrained global optimization approach, a sub-optimal control strategy could be obtained, which was more convenient to perform in practical fermentation, whilst a similar temperature profile and final glycerol yield to those of the optimal control strategy. The experimental and simulated results showed that, compared with those at the conventional temperature of 35 degreesC, the final glycerol yield at the sub-optimal temperature profile could be increased by 12-14% while the residual glucose was controlled at a value close to or less than 1% (w/v).