Diethyl phthalate (DEP) are classified as endocrine disruptors in water. In the present study, response surface methodology (RSM) was employed for flocculation process optimization in DEP removal from water. Two different copolymers, anionic polyacrylamide (APAM), were used as flocculants in this flocculation process including APAM1 and APAM2. APAM1 was polymerized by ultraviolet (UV) initiation, and APAM2 was polymerized without UV-initiation. The analysis result of variance demonstrated that the model was highly significant and reliable. Optimization by RSM with APAM1, the optimum conditions were dosage of 11.01 mg L−1, pH of 8.93, and stirring time of 6.29 min. And the optimum conditions with APAM2 were dosage of 13.68 mg L−1, initial pH of 8.73, and stirring time of 6.80 min. DEP removal efficiency of 83.97% was achieved by using flocculants APAM1 and 72.47% for APAM2. Scanning electron microscopy images and spectrum from nuclear magnetic resonance spectrometer (1H NMR) suggested that UV-initiation had played an important surface modification in APAM polymerization. In addition, the confirmation experiment results showed that the measured values had a good agreement with the predicted values, which demonstrated that RSM could be successfully used in flocculation process.