Gram-negative Serratia marcescens is a potential biological control agent of fungal plant diseases in many tropical regions. Its antifungal activity is due to the production of prodigiosin, protease, chitinase, and other substances that act to weaken the fungal cell wall and cause lysis. In the following investigation, a novel extracellular protein with antifungal activity against Rhizoctonia solani and Fusarium oxysporum was optimally produced, purified, and characterized from a native Serratia marcescens DT3. Under optimal conditions (medium containing 1.25% peptone, 0.5% yeast extract; temperature of 28 °C; initial pH of 7; culture time of 28 h), the antifungal activity of the extracellular extract was increased by a factor of 1.6-2.9 in comparison with the initial condition. The purified protein had an apparent molecular mass of 55 kDa. Its antifungal activity was retained at 80 °C for 30 min and after having been treated with proteinase K (0.5-4 μg/mL). The results of protein identification using a MALDI-TOF/TOF mass spectrometer suggested that the purified protein is indeed a serine 3-dehydrogenase.