Excessive glycerol obtained as a byproduct of biodiesel plants has been utilized increasingly as a versatile feedstock in biorefineries. Since the purchase cost of refined glycerol is more costly than crude glycerol, value-added chemicals such as dihydroxyacetone (DHA) should be produced from crude glycerol. Previously, a variety of naturally occurring microorganisms were proven capable of consuming crude glycerol, making microbial conversion more effective than the other approaches. This novel study, for the first time, simulates glycerol-based DHA production via microbial fermentation. The effects of carbon source types and glycerol types on DHA productivity are investigated. Simulated processes of DHA production are evaluated in terms of glycerol utilization, energy consumption, and economic and environmental aspects. This study reveals that the utilization of crude glycerol as the raw material can lead to the efficient production of DHA. The heat integration is also investigated in this work, which increases the total energy savings by 52–58%─the best production scenario yields an NPV of 656 million USD, a % IRR of 148.4%, and a payout period of 1.75 years. In addition, it is found that the microbial fermentation route is more cost-effective and less harmful to the environment than the catalytic oxidation route.