Electric field driven aggregation of oil droplets in oil-in-water (O/W) emulsion is a novel attempt for enhancing oil-water separation. The chaining and aggregation behavior of oil droplets in O/W emulsion under bidirectional pulsed electric field (BPEF) was studied in this work for the purpose of oil droplets separation from aqueous phase in O/W emulsion for the first time. The BPEF induced oil droplets to aggregate and take shape into three forms of congeries: oil-droplet chain, oil clusters and oil cluster chains. The aggregation effect of oil drops under BPEF voltage, frequency and duty cycle was explored by evaluating the size and formation time of congeries. It was discovered that BPEF voltage could enhance and accelerate oil droplets to aggregate but the aggregation effect was weakened by the increased BPEF frequency. And the best aggregation state of oil droplets under different duty cycles was achieved at BPEF duty cycle 70%. The demulsification performance of O/W emulsion under BPEF was investigated and its variation was consistent with the aggregation regularity and effect of oil drops in BPEF. Therefore, the microcosmic aggregation effect of oil drops in O/W emulsion under BPEF could be used to estimate the demulsification performance and separation effect of O/W emulsion.