An on-line detection device of trace moisture in operating transformer oil comprises an oil inlet, an oil-way pipeline, an oil outlet, a sealing ring, a trace moisture sensor, and a trace moisture detection unit, wherein the oil inlet is directly connected with a transformer oil sampling valve through an oil pump; the extracted oil contacts the trace moisture sensor through the oil-way pipeline to realize trace moisture measurement; the oil outlet is connected with a standby valve of the transformer to realize oil-way circulation; the sealing ring ensures the isolation of the oil-way pipeline from external air; signals sensed by the trace moisture sensor are transmitted to the trace moisture detection unit through cables. When the invention is used for on-line monitoring of trace moisture in transformer insulating oil, measuring errors caused by oil sample dead zones due to nonflowing oil samples are avoided.