The invention discloses a method for preparing a tetrodotoxin positive quality control sample. The method comprises the steps of selecting muscular tissues from fresh fugu rubripes, homogenizing, and measuring the content of tetrodotoxin in the muscular tissues to verify whether the muscular tissues contain the tetrodotoxin; if the muscular tissue is a positive sample, diluting the positive sample containing toxin with a negative sample and a dispersant, mixing uniformly, continuously measuring the content of the toxin in the sample, packaging the sample after the sample meets requirements, preserving at an appropriate temperature, and performing uniformity verification and stability analysis on the sample; if the muscular tissue is a negative sample, adding a tetrodotoxin standard solution into the negative samples, adding a dispersant, mixing uniformly, measuring the content of the tetrodotoxin in the sample, packaging the sample after the sample meets requirements, preserving at an appropriate temperature, and performing uniformity verification and stability analysis on the sample. The quality control sample is simple to operate, and has enough stability and uniformity and long preservation time, so that the quality control sample can be used for laboratory quality control.