Biofouling has become the focus of RO in water and wastewater treatment. Experimental investigation of RO biofouling mechanism and characteristics in wastewater advanced reuse was carried out by static test and cross-flow dynamic test on a lab-scale membrane fouling simulator(MFS).The experimental results show that the major contributors to biofouling are coli and filamentous bacteria; there usually is no biofouling on the RO membrane surface with the absence of microbial source and nutrient composition; it is easy to cause biofouling on the fouled membrane; the germicidal treatment and the protection in the shut-down period are important for RO. Guidance for the analysis and reduction of biofouling in the practical engineering is provided.