The invention discloses a liquid chromatography-mass spectrometry screening method of unknown toxic substances in blood. The liquid chromatography-mass spectrometry screening method comprises the following steps: taking a blood sample to be detected, adding acetonitrile precipitated protein, oscillating, centrifuging, taking a supernatant, filtering by using a 0.22-micron microporous organic filter membrane, then performing LC-MS/MS analysis, and judging whether the blood sample to be detected contains toxic substance components or not according to an analysis result; the used instrument is an ultraLC 100-XL-4000Q TRAP liquid chromatograph/mass spectrometer or an Agilent 1100 liquid chromatography-AB 3200 QTRAP triple serial quadrupole mass spectrometer from an AB company. By the liquid chromatography-mass spectrometry screening method, detection methods of different types of toxic substances are established, detection is fast and accurate, the detection limits of common toxic substances are given, high-sensitivity and effective detection of all target objects are guaranteed, a scientific data support is provided for a negative detection result, and thus a strong scientific basis is provided for relevant clinical diagnosis and rescue as well as criminal investigation and litigation.