The invention discloses a natural matrix standard sample containing 13 pesticide residues in tea and a preparation method thereof. Trifloxystrobin, boscalid, pyrimethanil, metalaxyl, bifenthrin, endosulfan, triadimefon, diniconazole, epoxiconazole, pyridaben, ametryn, napropamide, Acetochlor was sprayed on the tea trees or the soil of the test area, and the tea leaves were collected to make natural matrix standard samples. The tea natural matrix standard sample preparation method is scientific and reasonable, and has strong operability. The prepared sample has good uniformity, good stability, easy storage, and convenient use; the sampling volume, extraction, and purification methods are the same as those for daily analysis samples. The detection data obtained by different ambient temperatures and different detection personnel are reliable and accurate, suitable for use as a quality control method in detection work, and have high application and promotion value.