Since traditional Chinese medicine (TCM) is a complex mixture of multiple components, the application of methodologies for evaluating single-components Western medicine in TCM studies may have certain limitations. Appropriate strategies that recognize the integrality of TCM and connect to TCM theories remain to be developed. Here we use multiple unique approaches to study the scientific connotation of a TCM formula Dan-hong injection (DHI) without undermining its prescription integrity. The blood circulation improving and healing promoting effects of DHI were assessed by a qi stagnation blood stasis rat model and a mouse model of laser irradiation induced cerebral microvascular thrombosis. By UFLC-PDA-Triple Q-TOF-MS/MS and relevance analysis between chemical characters and biological effects, 82 chemical constituents and nine core components, whose blood circulation promoting effects were found comparable to that of whole DHI, were successfully identified. What’s more, the rationality of DHI prescription compatibility could be reflected not only in the maximum efficacy of the original ratio, but also in the interactions of compounds from different ingredient herbs, such as complementary activities and facilitating tissues distribution. This study provides scientific evidences in explanation of the clinical benefits of DHI, and also gives a good demonstration for the comprehensive evaluation of other TCM.