The invention discloses a brain malignant tumor cell combined quantitative detection card, a preparation method thereof, and a using method thereof. The brain malignant 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 collection zones, wherein the sample adding zone is positioned at one end of the microchannels, the bonding zones are connected with the one or more detection zones through the microchannels and the time valves and are positioned at the other end of the microchannels, and the detection zones are connected with the collection zones through the microchannels; a plurality of sample adding cavities are formed in the sample adding zone, the sample adding cavities are cylindrical microchannels, and the bottoms of the sample adding cavities are directly connected to the bonding zones. According to the brain malignant 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, brain malignant 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.