The biodegradation characteristics of dimethyl phthalate (DMP) by three freshwater unicellular organisms were investigated in this study. The findings revealed that all the organisms were capable of metabolizing DMP; among them, Cyanothece sp. PCC7822 achieved the highest degradation efficiency. Lower concentration of DMP supported the growth of the Cyanobacteria; however, with the increase of DMP concentration growth of Cyanobacteria was inhibited remarkably. Phthalic acid (PA) was detected to be an intermediate degradation product of DMP and accumulated in the culture solution. The optimal initial pH value for the degradation was detected to be 9.0, which mitigated the decrease of pH resulting from the production of PA. The optimum temperature for DMP degradation of the three species of organisms is 30°C. After 72 hours' incubation, no more than 11.8% of the residual of DMP aggregated in Cyanobacteria cells while majority of DMP remained in the medium. Moreover, esterase was induced by DMP and the activity kept increasing during the degradation process. This suggested that esterase could assist in the degradation of DMP.