The invention discloses a gynecological tumor cell combined quantitative detection card, a preparation method thereof, and a using method thereof. The gynecological tumor cell combined quantitative detection card comprises a bottom card and a surface card; the bottom card is provided with a sample adding zone, bonding zones, microchannels, time valves, detection zones, and waste liquid zones, wherein the sample adding zone is positioned at one end of the microchannels, the bonding zones are connected with the multiple detection zones through the microchannels and the time valves and are positioned at the other end of the microchannels, the waste liquid zones are connected to the sample adding zone through the microchannels, and the bottoms of the bonding zones and the detection zones are both parallelly connected with the microchannels; and the microchannels are connected with the time valves. According to the gynecological tumor cell combined quantitative detection card, the preparation method thereof, and the using method thereof,the flow rate and the flow quantity of a to-be-tested sample are controlled by the microfluidic technology, gynecological tumor cells with the same marker enter the detection zones through a microchannel, so that mutual interference is avoided, thus the error is reduced, and reaction sensitivity is increased; and the to-be-tested sample is prepared into water-in-water micro-droplets, so that the detection limit is increased.