The safety of total flavonoids of Penthorum chinense Pursh (TFPCP) was evaluated. An acute oral toxicity test, three genotoxicity tests, and a 28-day feeding study in rats were conducted to investigate the toxicological safety of TFPCP according to GB 15193-2014 Procedures for Toxicological Assessment of Food and Technical Guidelines for Toxicological Examination and Evaluation of Health Food and its Raw Materials (2020 Edition). These oral acute toxicity studies in mice revealed that the median lethal dose (LD50) of TFPCP was greater than 10 g/(kg•BW), suggesting that TFPCP is non-toxic. In the bacterial reverse mutation (Ames) test, all dose groups produced negative results. Additionally, for all dose groups, the colony counts did not exceed twice those of the solvent controls for TA97A, TA98, and TA100 or three times that for TA1535 (P>0.05). Gavage administration of TFPCP in mice at doses of 2.4, 1.2, and 0.6 g/(kg•BW) revealed no significant differences in mammalian erythrocyte micronucleus and chromosomal aberration test results in mouse spermatocytes compared with the negative control (P>0.05). Three different doses of TFPCP (2.4, 1.2, and 0.6 g/(kg•BW)) were administered to rats by gavage once daily for 28 consecutive days. No significant differences were observed between treated and control groups in terms of body weight, routine urinary and blood parameters, blood biochemical indices, and major organ coefficients (P>0.05). Similarly, histopathological examination of liver and kidney tissues revealed no significant pathological changes. Thus, TFPCP exhibited no acute toxicity, genotoxicity, or subacute toxicity under the conditions examined in this research and demonstrated excellent safety. These findings provide a scientific basis for development of TFPCP as a functional health food.