The invention discloses a kit for detecting pregnant woman peripheral blood free RNA screening 21-trisomy syndrome based on time-of-flight mass spectrometry. The kit comprises a PCR reaction system and a single base extension reaction system. The kit provided by the invention is combined with time mass spectrometry detection, and has the following advantages: 1, the detection accuracy is high; 2,the detection flux is high; 3, the detection sensitivity is high; 4, the detection sites are flexible and variable; 5, the detection process is simple, and the operation time is short; 6, the detection cost is low; 7, prenatal non-invasive detection is performed, damage to the pregnant woman is extremely small, psychological burdens of the pregnant woman are reduced, and a detection sample is peripheral blood of the pregnant woman and is easy to obtain; and 8, the whole detection operation is easy to repeat, mass spectrum chip sample application is fully automatic, the detection result is automatically analyzed through software, and errors caused by improper manual operation can be reduced.