The seed of Moringa oleifera (MO) is a well-known coagulant used in water and wastewater treatment, especially in developing countries. The main mechanism of MO seed extract in coagulation is the positive protein component for charge neutralization. The method for efficient extraction of MO seed is very important for high coagulation activity. In this study, the effects of extraction mixing speed and extraction time of MO on coagulation activity were evaluated using a distilled water extraction method. Although the rotation per minute for extraction did not affect the coagulation efficiency, the extraction time strongly affected the coagulation efficiency of the extract. To evaluate the characteristic change of MO extract by extraction time, the charge of MO extract and protein characteristic in MO extract were analysed. As the extraction time was short, more positive charge and higher protein content were observed. For detailed protein analysis, the fluorescence spectroscopic study (EEM analysis) was performed. The tryptophan-like peak increased at longer extraction times. For efficient extraction of MO seed, a short extraction time is strongly recommended.